Multi-strain population control system and method

文档序号：751315 发布日期：2021-04-02 浏览：22次中文

阅读说明：本技术 多菌株种群控制系统和方法 (Multi-strain population control system and method ) 是由 J·哈斯提 M·J·廖 M·O·丁于 2019-06-07 设计创作，主要内容包括：本文提供了一种多菌株种群控制系统、方法、试剂盒和组合物。还提供了用于在多菌株生态系统,以及使用同步裂解回路结合多种毒素/抗毒素系统在长时间内连续循环以瞬时排列多菌株生态系统或培养物中培养细菌细胞的方法、系统、试剂盒和组合物。(Provided herein are multi-strain ethnic group control systems, methods, kits, and compositions. Also provided are methods, systems, kits and compositions for culturing bacterial cells in a multi-strain ecosystem, and using a simultaneous lytic loop in conjunction with multiple toxin/antitoxin systems to continuously cycle over an extended period of time to transiently align the multi-strain ecosystem or culture.)

1. A method, comprising:

culturing a first bacterial strain in a growth environment for a first period of time;

adding a second bacterial strain to the growth environment and culturing the second bacterial strain for a second period of time;

adding a third bacterial strain to the growth environment and culturing the third bacterial strain for a third period of time;

Wherein each of the first, second and third strains comprises a toxin system;

wherein the toxin system of the first bacterial strain produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin;

wherein the toxin system of the second bacterial strain produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and

wherein the toxin system of the third bacterial strain produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin; and

optionally wherein each of the first, second and third bacterial strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule.

2. The method of claim 1, wherein the quorum sensing molecule is the same in each of the first, second, and third bacterial strains.

3. The method of any one of claims 1-2, wherein the toxin system of each of the first, second, third and one or more additional bacterial strains can be independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

4. The method of any one of claims 1-3, wherein in each of at least first, second, and third bacterial strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene.

5. A method according to any one of claims 1 to 4, wherein the lytic gene in each of at least the first, second and third bacterial strains is E from bacteriophage Φ X174.

6. The method of any one of claims 1-5, wherein the activatable promoter in each of at least the first, second, and third bacterial strains is a LuxR-AHL activatable luxI promoter and the activator gene is LuxI.

7. The method of any one of claims 1-6, wherein one or more of the first, second, and third bacterial strains encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

8. The method of claim 7, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

9. The method of claim 8, wherein the therapeutic agent is selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

10. A method, comprising:

providing n bacterial strains including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain;

co-culturing each bacterial strain in turn in a growth environment for one or more independent periods of time;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

wherein the toxin system of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain;

Wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

11. The method of claim 10, wherein the toxin systems of the n bacterial strains are independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

12. The method of any one of claims 10-11, wherein one or more time periods partially or completely overlap.

13. The method of any one of claims 10-12, wherein one or more of the n bacterial strains encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

14. The method of claim 13, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

15. The method of claim 14, wherein the therapeutic agent is selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

16. The method of any one of claims 10-15, wherein each of the second through nth bacterial strains does not produce the second toxin of the toxin system of the previous strain, and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain.

17. A bacterial strain comprising a toxin system, wherein the toxin system produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the bacterial strain does not produce the second toxin.

18. The bacterial strain of claim 17, wherein the toxin system is encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

19. The bacterial strain of any one of claims 17-18, wherein the bacterial strain further comprises a nucleic acid encoding a therapeutic agent.

20. The bacterial strain of claim 19, wherein said therapeutic agent is selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

21. The bacterial strain of any one of claims 19-20, wherein the therapeutic agent is a therapeutic polypeptide.

22. The bacterial strain of any one of claims 19-21, wherein the therapeutic agent is cytotoxic or cytostatic to a target cell.

23. A pharmaceutical composition comprising any one or more of the bacterial strains of claims 17-22.

24. A system, comprising:

n bacterial strains including at least a first bacterial strain, a second bacterial strain and an nth bacterial strain;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

25. The system of claim 24, wherein n is 3.

26. The system of claim 24, wherein n is 4, 5, 6, 7, 8, 9, or 10.

27. A method for treating a disease in a subject in need thereof, comprising:

administering to a subject a therapeutically effective amount of each of n bacterial strains, the n bacterial strains including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain;

Wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

28. The method of claim 27, wherein n is 3.

29. The method of claim 27, wherein n is 4, 5, 6, 7, 8, 9, or 10.

30. The method of any one of claims 27-29, wherein each of the toxin systems is independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

31. The method of any one of claims 27-30, wherein each of the second through nth bacterial strains does not produce the second toxin of the toxin system of the previous strain, and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain.

32. The method of any one of claims 27-31, wherein the disease is cancer or an infection.

33. The method of claim 32, wherein the infection is caused by an infectious agent selected from the group consisting of: campylobacter jejuni (Campylobacter jejuni), Clostridium botulinum (Clostridium botulinium), Escherichia coli (Escherichia coli), Listeria monocytogenes (Listeria monocytogenes), and Salmonella (Salmonella).

34. The method of claim 33, wherein the cancer is selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular carcinoma, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer.

Technical Field

The present invention relates to methods, systems, kits and compositions for culturing bacterial cells, and more particularly, to methods, systems, kits and compositions for multi-strain ecosystems, including population-regulated multi-lytic systems.

Background

Microbial ecologists are increasingly inclined to integrate small synthetic ecosystems ^1–5Exploring the complexity of the natural microbiome as a reduction theory tool^6,7. At the same time, synthetic biologists have taken the loop from a single-cell gene^8–11Steering to control the entire population using intercellular signal transduction^12–16。

Disclosure of Invention

Without wishing to be bound by theory, the present inventors disclose a triple or multi-lytic system that can be used to engineer multi-strain ecosystems and/or achieve cyclic delivery of one or more payloads (e.g., therapeutic agents). For example, each strain can deliver a payload of one or more therapeutic proteins while excluding previously administered strains or preexisting strains in the site of disease. For example, there may be at least three strains that make up the system, where each strain produces one toxin (killing one of the other two strains) and one antitoxin that protects itself, while the other strains are not targeted by toxins. For example, in an exemplary system comprising strains labeled as strain a, strain B, and strain C, strain a can produce a toxin that kills strain B; strain B in turn can produce a toxin that kills strain C, which in turn can produce a toxin that kills strain a. More strains may be present in the system, and may or may not participate in the circulating toxin system. The system is not limited to a three strain system, which may consist of more than three strains (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 strains), wherein each strain produces one toxin and is capable of killing another strain in the system that does not have an antitoxin, thereby allowing for a circulatory system (consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12 or more strains). Furthermore, the system may be composed of more than three strains (e.g. 4, 5, 6, 7, 8, 9, 10, 11 or 12 strains), wherein each of the at least three strains produces one toxin and is capable of killing another strain in the system that does not have an antitoxin, thereby allowing a circulatory system to be formed between at least three of the strains, while the other strains of the system are not controlled according to the three or more lysis loops. In one aspect, the system can provide for specific delivery of a therapeutic agent via an engineered strain over a period of time until a subsequent strain in the system is administered, thereby spatially and temporally segregating delivery of the specific therapeutic agent as desired.

In some embodiments, each strain can produce a therapeutic protein or a set of therapeutic proteins, the expression of which can be controlled by a genetic circuit such as a Synchronous Lytic Circuit (SLC). In some embodiments, exemplary SLCs may include those described in International application PCT/US18/33555, which is incorporated herein by reference in its entirety.

In some embodiments, the triple-or multi-lytic system utilizes a well-characterized E.coli-specific toxin-antitoxin system in combination with a Lux-AHL quorum-sensing lysis loop to not only provide self-limiting control of bacterial population size, but also to enable precise removal of unwanted strains in a cyclic "stone-cloth-scissors" fashion for long-term continuous culture. A triple or multi lysis system may provide specific delivery of a therapeutic protein via an engineered strain over a period of time until a subsequent strain in the system is administered, thereby spatially and temporally isolating delivery of the specific therapeutic protein. The triple or multiple lytic system may also be engineered to export a specific profile of the therapeutic protein to the disease site.

In another aspect, triple or multi-split systems may also be suitable for engineering "split" ecosystems, where multiple strains may metabolize or produce certain intermediates with the goal of synthesizing compounds of commercial value. Other similar applications-triple or multi-lysis systems may include repair and sensing applications.

Furthermore, the use of a triple or multiple lysis system as a means of ensuring stability of the circuit is not necessarily limited to therapeutic applications, and may be applicable to any application requiring stability of a genetic construct in a bacterium.

Provided herein are methods of maintaining a co-culture by quorum sensing, comprising: co-culturing at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10) strains at a ratio (e.g., 1:1:1) over a period of time; wherein each of the at least three strains comprises a lytic plasmid and an activator plasmid.

Also provided herein are methods of maintaining a co-culture by quorum sensing, comprising: co-culturing at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10) strains at a ratio (e.g., 1:1:1) over a period of time; wherein each of the at least three strains comprises a toxin system.

Also provided herein are methods of maintaining a co-culture comprising: co-culturing at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10) strains at a ratio (e.g., 1:1:1) over a period of time; wherein each of the at least three strains comprises a toxin system.

In some embodiments, the at least three bacterial strains are escherichia coli (e.coli), salmonella typhimurium (s.typhimurium), or bacterial variants thereof. In some embodiments, the at least three bacterial strains are gram-negative bacterial strains, such as Salmonella (Salmonella), Acetobacter (Acetobacter), Enterobacter (Enterobacter), clostridium (Fusobacterium), Helicobacter (Helicobacter), Klebsiella (Klebsiella), or escherichia coli (e.coli) strains. In some embodiments, the at least three bacterial strains are gram-positive bacterial strains, such as actinomycete (actinomycete) strains, Bacillus (Bacillus) strains, Clostridium (Clostridium) strains, Enterococcus (Enterococcus) strains, or Lactobacillus (Lactobacillus) strains. In some embodiments, the at least three bacterial strains are all gram-negative bacterial strains or all gram-positive bacterial strains. In some embodiments, at least one of the at least three bacterial strains is a gram-negative bacterial strain. In some embodiments, at least one of the at least three bacterial strains is a gram-positive bacterial strain.

In some embodiments, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene. In some embodiments, the lytic gene is E from bacteriophage Φ X174. In some embodiments, the activatable promoter is a LuxR-N-Acyl Homoserine Lactone (AHL) activatable luxI promoter, and the activator gene is luxI. In some embodiments, the activatable promoter is a RpaR-N-Acyl Homoserine Lactone (AHL) activatable RpaI promoter and the activator gene is RpaI. In some embodiments, the reporter gene is Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), or a variant thereof. In some embodiments, the degradation tag is an ssrA-LAA degradation tag. In some embodiments, the at least three bacterial strains each comprise a lytic plasmid and an activator plasmid. In some embodiments, each of the at least three bacterial strains comprises a different reporter gene.

In some embodiments, the co-culture is inoculated at a ratio of 1:1: 1. In some embodiments, one or more bacterial strains may be co-cultured. In some embodiments, the co-culture is inoculated at a specific ratio (e.g., 1:1, 1:5, 1:10, 1:1:1, 1:5:1, etc.) based on the strain used, the desired characteristics of the system, the outcome goal, etc. In some embodiments, one or more strains in the system may have a growth advantage over one or more other strains in the system.

In some embodiments, the plasmid is integrated into the genome of at least one of the at least three bacterial strains.

In some embodiments, the co-culturing is performed in a microfluidic device. In some embodiments, the co-culturing is performed in a cell culture vessel (e.g., cell culture plate, bioreactor).

In some embodiments, the period of time is 0 to 72 hours (e.g., 0 to 72, 0 to 60 hours, 0 to 48 hours, 0 to 36 hours, 0 to 24 hours, 0 to 16 hours, 0 to 14 hours, 0 to 12 hours, 0 to 10 hours, 0 to 8 hours, 0 to 6 hours, 0 to 4 hours, 0 to 2 hours, 2 to 72 hours, 2 to 60 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 72 hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 16 hours, 4 to 14 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 8 hours, 6 to 10 hours, 6 to 12 hours, 6 to 14 hours, 6 to 16 hours, 6 to 22 hours, 6 to 6 hours, 6 to 8 hours, 6 to 10 hours, 6 to 12 hours, 6 to 14 hours, 6 hours When the current is over; 8 to 10 hours; 8 to 12 hours; 8 to 16 hours; 8 to 24 hours; 8 to 36 hours; 8 to 48 hours; 8 to 60 hours; 8 to 72 hours; 1 to 2 hours; 1 to 3 hours; 1 to 4 hours; 1 to 6 hours; 1 to 8 hours; 1 to 10 hours; 1 to 12 hours; 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours).

In some embodiments, the co-culture of the at least three bacterial strains is in a constant lytic state; wherein the constant lytic state is characterized by a steady state balance of growth and lysis of the at least three bacterial strains.

Provided herein are bacterial strains comprising a lytic plasmid and an activator plasmid; wherein the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene.

In some embodiments, the lytic gene is E from bacteriophage Φ X174.

In some embodiments, the activatable promoter is a LuxR-N-Acyl Homoserine Lactone (AHL) activatable luxI promoter, and the activator gene is luxI.

Also provided herein are pharmaceutical compositions comprising any of the bacterial strains described herein. In some embodiments, the pharmaceutical composition is formulated for in situ drug delivery. The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral or parenteral administration, such as intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal and rectal administration. As used herein, the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.

Provided herein is a system comprising: co-culture of at least three bacterial strains as described herein.

Provided herein are drug delivery systems including any of the systems described herein. Provided herein are periodic drug delivery systems including any of the systems described herein.

Provided herein is a method of treating a disease in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of at least three bacterial strains as described herein or a pharmaceutical composition as described herein to treat a disease in the subject. In some embodiments, the administering comprises administering each of the at least three bacterial strains to the subject sequentially. In some embodiments, the administering comprises administering each of the at least three bacterial strains to the subject simultaneously. In some embodiments, each of the at least three bacterial strains expresses a different therapeutic agent. In some embodiments, the disease is cancer or infection.

Provided herein are microfluidic sample wells (traps) comprising any of the systems described herein.

Provided herein are microfluidic devices comprising one or more microfluidic sample wells. In some embodiments, the microfluidic system further comprises at least one channel in fluid communication with the microfluidic sample well.

Provided herein is a method comprising culturing a first bacterial strain in a growth environment for a first time period, adding a second bacterial strain to the growth environment and culturing the second bacterial strain for a second time period, adding a third strain to the growth environment and culturing the third strain for a third time period, wherein each of the first, second, and third strains comprises a toxin system, wherein the toxin system of the first strain produces a first toxin/first antitoxin pair and a third antitoxin from the third toxin/third antitoxin pair, wherein the first strain does not produce the third toxin, wherein the toxin system of the second strain produces the second toxin/second antitoxin pair and the first antitoxin from the first toxin/first antitoxin pair, wherein the second strain does not produce the first toxin, wherein the toxin system of the third bacterial strain produces the third toxin/third antitoxin pair and the second antitoxin from the second toxin/second antitoxin pair A second toxin, wherein the second toxin is not produced by the second bacterial strain, and optionally wherein each of the first, second and third bacterial strains comprises a lytic plasmid having a lytic gene under the control of an activatable promoter, and an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules, wherein both the activatable promoter of the lytic gene and expression of the activator gene are activatable by the quorum sensing molecule.

Implementations may include one or more of the following features. The quorum sensing molecule may be different in each of the first, second and third bacterial strains. The quorum sensing molecules of each of the first, second and third strains each have no or substantially no effect on the activatable promoter of the lytic gene of the other strain. The quorum sensing molecule may be the same in each of the first, second and third bacterial strains. The method may further comprise culturing or co-culturing one or more additional bacterial strains in the growth environment. Each of the one or more additional bacterial strains may include a lytic plasmid having a lytic gene under the control of an activatable promoter and an activator plasmid having an activator gene, the expression of which promotes accumulation of the quorum sensing molecule, wherein both the activatable promoter of the lytic gene and the expression of the activator gene are activatable by the quorum sensing molecule. The quorum sensing molecule may be different in each of the first, second, and third bacterial strains and the one or more additional bacterial strains. The quorum sensing molecule of each of the first, second and third strains and the one or more additional bacterial strains each has no or substantially no effect on an activatable promoter of a lytic gene of another strain used in the growth environment. The quorum sensing molecule may be the same in each of the first, second, and third bacterial strains and the one or more additional bacterial strains. Each of the one or more additional bacterial strains may include a toxin system. The toxin system of each of the first, second, third and one or more additional strains may be independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof. The lytic plasmid and the activator plasmid of each of the at least first, second and third strains may be copies of the same plasmid. The lytic plasmid and the activator plasmid of each of the at least first, second and third strains may be different plasmids. At least the first, second and third bacterial strains are metabolically competitive. At least the first, second and third strains may be selected from escherichia coli, salmonella typhimurium or bacterial variants thereof. Each of the at least first, second and third strains cannot have a growth advantage over another strain in the growth environment. In each of the first, second and third strains, the lytic plasmid may comprise a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene. The lytic gene in each of the at least first, second and third strains may be E from the bacteriophage Φ X174. The activatable promoter in each of the at least first, second and third bacterial strains may be a LuxR-AHL activatable luxI promoter and the activator gene may be luxI. At least one reporter gene is selected from the group consisting of genes encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP) or variants thereof. The degradation tag may be an ssrA-LAA degradation tag. At least one of the plasmids may be integrated into the genome of at least one of the first, second and third bacterial strains. The culturing may be performed in a microfluidic device. The culturing may be performed in a bioreactor. The culturing may be performed in vivo. Each of the first, second, and third time periods may vary from about 12 hours to about 72 hours. Each of the first, second, and third time periods may be selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours. The first, second and third time periods may be selected from 12 hours, 24 hours, 48 hours, 72 hours and 96 hours. Each of the first, second, and third time periods may vary from about 1 hour to about 72 hours. Each of the first, second, and third time periods may vary from about 1 hour to about 48 hours. Each of the first, second and third time periods may vary from about 1 hour to about 24 hours. Each of the first, second, and third time periods may vary from about 1 hour to about 18 hours. Each of the first, second, and third time periods may vary from about 1 hour to about 12 hours. Each of the first, second, and third time periods may vary from about 1 hour to about 6 hours. Each of the first, second, and third time periods may vary from about 1 hour to about 3 hours. The first, second and third time periods may occur sequentially. The first and second, second and third, or first and third time periods may partially or completely overlap. One or more of the first, second and third bacterial strains may encode a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter. The promoter may be an activatable promoter. The promoter may be activated by a quorum sensing molecule. The promoter may be a constitutive promoter. The heterologous nucleic acid and/or heterologous protein can be a therapeutic agent. The therapeutic agent may be selected from: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof. The inhibitory nucleic acid may be an siRNA, shRNA, miRNA or antisense nucleic acid.

Also provided herein is a method comprising providing n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, each bacterial strain being co-cultured in a growth environment at separate time periods in sequence, wherein each of the second bacterial strain through the nth bacterial strain has a previous bacterial strain, where n can be at least 3, wherein each of the n bacterial strains includes a lytic plasmid having a lytic gene under control of an activatable promoter and an activator plasmid having an activator gene whose expression promotes accumulation of a quorum sensing molecule, wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activatable by the quorum sensing molecule, wherein each of the n bacterial strains includes a toxin system, wherein the toxin system of each of the second bacterial strain through the nth bacterial strain independently produces the toxin system of the second bacterial strain through the nth bacterial strain A first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein a toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by an nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Implementations may include one or more of the following features. In each of the n strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene. The lytic genes of the n strains are E of phi X174 phage. The activatable promoter in each of the n bacterial strains may be a LuxR-AHL activatable luxI promoter and the activator gene may be luxI. At least one reporter gene is selected from the group consisting of genes encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP) or variants thereof. The degradation tag may be an ssrA-LAA degradation tag. At least one of the plasmids may be integrated into the genome of at least one of the n bacterial strains. Quorum sensing molecules may be different in each of the n bacterial strains. Wherein the quorum sensing molecule of each of the n strains has no or substantially no effect on the activatable promoter of the lytic gene of the other strain. The quorum sensing molecule can be the same in each of the n bacterial strains. The lytic and activator plasmids of each of the n strains may be copies of the same plasmid. The lytic and activator plasmids of each of the n strains may be different plasmids.

Also provided herein is a method comprising providing n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain; sequentially co-culturing each bacterial strain in a growth environment for a separate period of time, wherein each of the second through nth bacterial strains has a previous bacterial strain, wherein n may be at least 3, wherein each of said n bacterial strains comprises a toxin system, wherein the toxin system of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against a previous bacterial strain, wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain. The n bacterial strains are metabolically competitive. Each of the n bacterial strains may be selected from escherichia coli, salmonella typhimurium or bacterial variants thereof. In a growing environment, each of the n bacterial strains is unlikely to have a growth advantage over the other. The toxin systems of the n bacterial strains may be independently encoded in the genome, the plasmid, multiple plasmids, or a combination thereof. The culturing may be performed in a microfluidic device. The culturing may be performed in a bioreactor. The culturing may be performed in vivo. Each of the separate time periods may vary from about 12 hours to about 72 hours. Each of the separate time periods may be selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours. Each of the independent time periods may be selected from 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours. Each time period can vary independently from about 1 hour to about 72 hours. Each time period can vary independently from about 1 hour to about 48 hours. Each time period can vary independently from about 1 hour to about 24 hours. Each time period can vary independently from about 1 hour to about 18 hours. Each time period can vary independently from about 1 hour to about 12 hours. Each time period can vary independently from about 1 hour to about 6 hours. Each time period can vary independently from about 1 hour to about 3 hours. One or more time periods may partially or completely overlap. One or more of the n bacterial strains may encode a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter. The promoter may be an activatable promoter. The activatable promoter may be activated by a quorum sensing molecule. The promoter may be a constitutive promoter. The heterologous nucleic acid and/or heterologous protein can be a therapeutic agent. The therapeutic agent may be selected from: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof. The inhibitory nucleic acid may be an siRNA, shRNA, miRNA or antisense nucleic acid. Each of the second to nth bacterial strains is incapable of producing the second toxin of the toxin system of the previous strain, and the first bacterial strain is incapable of producing the fourth toxin of the toxin system of the nth bacterial strain.

Also provided herein is a bacterial strain comprising a lytic plasmid and an activator plasmid, wherein the lytic plasmid comprises a lytic gene, an activatable promoter, and an optional reporter gene, and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene, wherein the bacterial strain further comprises a toxin system, wherein the toxin system produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the bacterial strain does not produce the second toxin.

Implementations may include one or more of the following features. The lytic gene may be E from phage Φ X174. The activatable promoter may be a LuxR-AHL activatable luxI promoter and the activator gene may be LuxI. An activator gene may encode a molecule that directly or indirectly activates an activatable promoter. The molecule that directly or indirectly activates the activatable promoter may be a quorum sensing molecule. The lytic gene is operably linked to an activatable promoter. The reporter gene on the lytic plasmid is operably linked to an activatable promoter. The activator plasmid can further comprise an activatable promoter. The activatable promoter of the activator plasmid may be a copy of the activatable promoter of the lytic plasmid. The activator gene is operably linked to the activatable promoter of the activator plasmid. The reporter gene of the activator plasmid is operably linked to the activatable promoter of the activator plasmid. The degradation tag is operably linked to the activatable promoter of the activator plasmid. The toxin system is operably linked to an activatable promoter of the lytic plasmid. The degradation tag may be an ssrA-LAA degradation tag. The reporter gene of the lytic plasmid, the reporter gene of the activator plasmid, or both may be a fluorescent protein. The reporter gene of the lytic plasmid and the reporter gene of the activator plasmid may be different genes.

Also provided herein are bacterial strains comprising a toxin system, wherein the toxin system produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the bacterial strains do not produce the second toxin.

Implementations may include one or more of the following features. The bacterial strain may encode a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter. The promoter may be an activatable promoter. The activatable promoter may be activated by a quorum sensing molecule. The promoter may be a constitutive promoter. The heterologous nucleic acid and/or heterologous protein can be a therapeutic agent. The toxin system may be encoded in a genome, a plasmid, a plurality of plasmids, or a combination thereof. The bacterial strain may also include a nucleic acid encoding a therapeutic agent. The therapeutic agent may be selected from: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof. The therapeutic agent may be a therapeutic polypeptide. The therapeutic agent may have a cytotoxic or cytostatic effect on the target cells. The target cell may be a cancer cell or an infected cell.

Also provided herein are pharmaceutical compositions comprising any one or more of the bacterial strains described herein. The pharmaceutical composition can be formulated for in situ drug delivery.

Also provided herein is a system comprising a first bacterial strain comprising a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene, and the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene, wherein the first bacterial strain further comprises a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin; the second bacterial strain comprises a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene, wherein the second bacterial strain further comprises a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and a third bacterial strain comprising a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene, the third activator plasmid comprising a third activator gene, optionally a third degradation tag, and optionally a third reporter gene, wherein the third bacterial strain further comprises a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Also provided herein is a system comprising a first bacterial strain comprising a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin, wherein the first bacterial strain further optionally comprises a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and an optional first reporter gene, and the first activator plasmid comprises a first activator gene, an optional first degradation tag, and an optional first reporter gene; a second bacterial strain comprising a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain further optionally comprises a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and a third bacterial strain comprising a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the third bacterial strain can further optionally comprise a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene, and the third activator plasmid comprises a third activator gene, optionally a third degradation tag, and optionally a third reporter gene.

Implementations of some systems may include one or more of the following features. The system may also include one or more additional bacterial strains. The first, second, and third toxin systems may be independently encoded in a genome, a plasmid, a plurality of plasmids, or a combination thereof.

Also provided herein is a system comprising n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second through nth bacterial strains has a previous strain, wherein n can be at least 3, wherein each of the n strains includes a toxin system, wherein the toxin system of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from the second toxin/second antitoxin pair produced by the previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from the fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Also provided herein is a system comprising providing n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second bacterial strain through the nth bacterial strain has a previous bacterial strain, wherein n can be at least 3, wherein each of the n bacterial strains includes a lytic plasmid having a lytic gene under the control of an activatable promoter and an activator plasmid having an activator gene, the expression of which promotes accumulation of quorum sensing molecules, wherein both the activatable promoter of the lytic gene and the expression of the activator gene can be activated by the quorum sensing molecules, wherein each of the n bacterial strains includes a toxin system, wherein the toxin system of each of the second bacterial strain through the nth bacterial strain independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by an nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Implementations of some systems may include one or more of the following features. The value of n may be 3. The value of n may be 4, 5, 6, 7, 8, 9 or 10. Each of the toxin systems may be independently encoded in a genome, a plasmid, multiple plasmids, or a combination thereof. Each of the second to nth bacterial strains is incapable of producing the second toxin of the toxin system of the previous strain, and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain.

Also provided herein is a kit comprising a first pharmaceutical composition comprising a first bacterial strain comprising a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene, and the first activator plasmid comprises a first activator gene, a first degradation tag, and optionally a first reporter gene, wherein the first bacterial strain further comprises a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce a third toxin; a second pharmaceutical composition comprising a second bacterial strain comprising a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, a second degradation tag, and optionally a second reporter gene, wherein the second bacterial strain further comprises a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and a third pharmaceutical composition comprising a third bacterial strain comprising a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene, the third activator plasmid comprising a third activator gene, a third degradation tag, and optionally a third reporter gene, wherein the third bacterial strain further comprises a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Also provided herein is a kit comprising a first pharmaceutical composition comprising a first bacterial strain comprising a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin, wherein the first bacterial strain further optionally comprises a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene, and the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene; a second pharmaceutical composition comprising a second bacterial strain comprising a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain further optionally comprises a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and an optional second reporter gene, the second activator plasmid comprising a second activator gene, an optional second degradation tag, and an optional second reporter gene; and a third pharmaceutical composition comprising a third bacterial strain comprising a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the third bacterial strain further optionally comprises a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and an optional third reporter gene, and the third activator plasmid comprises a third activator gene, an optional third degradation tag, and an optional third reporter gene.

Some kit embodiments may include one or more of the following features. The first, second, and third toxin systems may be independently encoded in a genome, a plasmid, a plurality of plasmids, or a combination thereof. The kit may further comprise one or more additional bacterial strains in the first, second, third and/or one or more additional pharmaceutical compositions.

Also provided herein is a kit comprising n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second through nth bacterial strains has a previous strain, wherein n can be at least 3, wherein each of the n strains includes a toxin system, wherein the toxin system of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from the second toxin/second antitoxin pair produced by the previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin of the fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Also provided herein is a kit comprising providing n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second bacterial strain through the nth bacterial strain has a previous bacterial strain, wherein n can be at least 3, wherein each of the n bacterial strains comprises a lytic plasmid having a lytic gene under the control of an activatable promoter and an activator plasmid having an activator gene, the expression of which promotes the accumulation of quorum sensing molecules, wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activatable by the quorum sensing molecule, wherein the lytic plasmid of each of the n bacterial strains includes a toxin system, wherein the toxin system of the lytic plasmid of each of the second bacterial strain through the nth bacterial strain independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the first bacterial strain cleaves the toxin system of the plasmid to produce a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by an nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Some kit embodiments may include one or more of the following features. The value of n may be 3. The value of n may be 4, 5, 6, 7, 8, 9 or 10.

Also provided herein is a drug delivery system, including any one or more of the systems or kits provided herein.

Also provided herein is a periodic drug delivery system, including any one or more of the systems or kits provided herein.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of the following bacterial strains: a first bacterial strain comprising a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene, and the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene, wherein the first bacterial strain further comprises a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce a third toxin; a second bacterial strain comprising a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene, wherein the second bacterial strain further comprises a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and a third bacterial strain comprising a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene, the third activator plasmid comprising a third activator gene, optionally a third degradation tag, and optionally a third reporter gene, wherein the third bacterial strain further comprises a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of: a first medicament comprising a first bacterial strain comprising a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene, and the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene, wherein the first bacterial strain further comprises a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce a third toxin; a second drug comprising a second bacterial strain comprising a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene, wherein the second bacterial strain further comprises a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and a third drug comprising a third bacterial strain comprising a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene, the third activator plasmid comprising a third activator gene, optionally a third degradation tag, and optionally a third reporter gene, wherein the third bacterial strain further comprises a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of: a first bacterial strain comprising a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin, wherein the first bacterial strain further optionally comprises a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene, and the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene; a second bacterial strain comprising a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain further optionally comprises a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and a third bacterial strain comprising a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, and optionally further comprising a third lytic plasmid comprising a third lytic gene, a third activatable promoter, and optionally a third reporter gene, and a third activator plasmid comprising a third activator gene, optionally a third degradation tag, and optionally a third reporter gene.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of: a first pharmaceutical composition comprising a first bacterial strain comprising a first toxin system, wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin, wherein the first bacterial strain further optionally comprises a first lytic plasmid and a first activator plasmid, wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and an optional first reporter gene, and the first activator plasmid comprises a first activator gene, an optional first degradation tag, and an optional first reporter gene; also included are second pharmaceutical compositions comprising a second bacterial strain comprising a second toxin system, wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain further optionally comprises a second lytic plasmid and a second activator plasmid, wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene, and the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and a third pharmaceutical composition comprising a third bacterial strain comprising a third toxin system, wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, and optionally further comprising a third lytic plasmid and a third activator plasmid, wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene, and the third activator plasmid comprises a third activator gene, optionally a third degradation tag, and optionally a third reporter gene.

Implementations of some methods may include one or more of the following features. The administering can include administering to the subject the first, second, and third bacterial strains or each of the first, second, and third pharmaceutical compositions in sequence. The administering can include administering each of the first, second, and third pharmaceutical compositions simultaneously, and wherein each of the first, second, and third pharmaceutical compositions has a different release profile that releases the first, second, and third bacterial strains. Each of the first, second and third bacterial strains may express a different therapeutic agent.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second through nth bacterial strains has a previous bacterial strain, where n can be at least 3, wherein each of the n bacterial strains comprises a lytic plasmid having a lytic gene under the control of an activatable promoter and an activator plasmid having an activator gene whose expression promotes accumulation of a quorum sensing molecule, wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activatable by the quorum sensing molecule, wherein the lytic plasmid of each of the n bacterial strains comprises a toxin system, wherein the toxin system of the lytic plasmid of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the toxin system of the lytic plasmid of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Implementations of some methods may include one or more of the following features. The value of n may be 3. The value of n may be 4, 5, 6, 7, 8, 9 or 10.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of m pharmaceutical compositions, each pharmaceutical composition comprising at least one of n bacterial strains, the n bacterial strains comprising at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second through nth bacterial strains has a previous strain, wherein n can be at least 3, wherein each of the n strains comprises a toxin system, wherein the toxin system of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by the previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin of a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of m pharmaceutical compositions, wherein each pharmaceutical composition comprises at least one of n bacterial strains, wherein the n bacterial strains comprise at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain, wherein each of the second through nth bacterial strains has a previous bacterial strain, wherein n can be at least 3, wherein each of the n bacterial strains comprises a lytic plasmid having a lytic gene under the control of an activatable promoter and an activator plasmid having an activator gene, the expression of which promotes accumulation of quorum sensing molecules, wherein both the activatable promoter of the lytic gene and the expression of the activator gene can be activated by the quorum sensing molecule, wherein the lytic plasmid of each of the n bacterial strains comprises a toxin system, wherein the toxin system of the lytic plasmid of each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain, wherein the toxin system of the first bacterial strain lytic plasmid produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Implementations of some methods may include one or more of the following features. The value of n may be 3. The value of n may be 4, 5, 6, 7, 8, 9 or 10. The value m is n. Each of the toxin systems may be independently encoded in a genome, a plasmid, multiple plasmids, or a combination thereof. Each of the second to nth bacterial strains does not produce the second toxin of the toxin system of the previous strain, and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain. Administration may comprise administering to the subject n bacterial strains or each of the m pharmaceutical compositions in sequence. The administering may include administering each of the m pharmaceutical compositions simultaneously, wherein each of the m pharmaceutical compositions has a different release profile that releases the first, second, and third bacterial strains. Each of the first, second and third bacterial strains may express a different therapeutic agent. The disease may be cancer or infection. The infection may be caused by an infectious agent selected from the group consisting of: campylobacter jejuni (Campylobacter jejuni), Clostridium botulinum (Clostridium botulinium), Escherichia coli (Escherichia coli), Listeria monocytogenes (Listeria monocytogenes), and Salmonella (Salmonella). The cancer may be selected from: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular carcinoma, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer.

In some embodiments of any one or more of the methods, bacterial strains, pharmaceutical compositions, systems, or kits described herein, the one or more bacterial strains can express a therapeutic agent. In some embodiments of any one or more of the methods, bacterial strains, pharmaceutical compositions, systems, or kits described herein, the one or more bacterial strains may express a therapeutic agent selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

Also provided herein is a method comprising culturing a first bacterial strain in a growth environment for a first period of time, adding a second bacterial strain to the growth environment and culturing the second bacterial strain for a second period of time, adding a third bacterial strain to the growth environment and culturing the third bacterial strain for the second period of time, wherein each of the first, second and third bacterial strains comprises a toxin system, wherein the toxin system of the first bacterial strain produces a first toxin/first antitoxin pair and a third antitoxin from the third toxin/third antitoxin pair, wherein the first strain does not produce the third toxin, wherein the toxin system of the second bacterial strain produces the second toxin/second antitoxin pair and the first antitoxin from the first toxin/first antitoxin, wherein the second bacterial strain does not produce the first toxin, and wherein the toxin system of the third bacterial strain produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the toxin system may be encoded on a plasmid, on multiple plasmids, integrated into the host genome, or a combination thereof.

Implementations may include one or more of the following features. Each of the first, second, and third bacterial strains may comprise: a lytic plasmid having a lytic gene under the control of an activatable promoter; and an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules; wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule; wherein the quorum sensing molecules in each of the first, second, and third bacterial strains may be the same or different, wherein each quorum sensing molecule of each of the first, second, and third bacterial strains may or may not have any effect on the activatable promoter of the lytic gene of the other strain. Each lytic plasmid of the first, second and third bacterial strains may be a plasmid containing the toxin system of said bacterial strain, or wherein each activator plasmid of said first, second and third bacterial strains may be a plasmid containing the toxin system of said bacterial strain, or wherein the toxin system of each bacterial strain may be integrated into the genome.

Also provided herein is a method of culturing n bacterial strains, comprising culturing a first bacterial strain of the n bacterial strains in a culture for a first period of time, adding a second bacterial strain of the n bacterial strains to the culture and culturing the second bacterial strain for a second period of time, adding each remaining bacterial strain of the n bacterial strains to the culture and culturing each bacterial strain for a period of time, wherein n is a number of 3 or more, wherein the plasmid of each of the n bacterial strains comprises a toxin system, wherein the toxin system of the first bacterial strain produces a first toxin/first antitoxin pair and an nth antitoxin from the nth toxin/nth antitoxin pair, wherein the first strain does not produce the nth toxin, wherein the toxin systems of the plasmids of the mth of the other bacterial strains of the n bacterial strains produce the mth toxin/mth antitoxin pair and from the (m-1) Toxin/(m-1) th antitoxin of (m-1) th antitoxin, where m may be an element of group {2,3, …, n }.

Embodiments of the methods described herein can include one or more of the following features. Each of the n bacterial strains may include: a lytic plasmid having a lytic gene under the control of an activatable promoter; and an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules; wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule; wherein the quorum sensing molecules in each of the n bacterial strains may be the same or different, wherein each quorum sensing molecule of each of the n bacterial strains may or may not have any effect on the activatable promoter of the lytic gene of another of the n bacterial strains. Each lytic plasmid of the n bacterial strains may be a plasmid containing the toxin system of the bacterial strain. Each of the n bacterial strains is capable of producing a payload. Each of the n bacterial strains is capable of producing a different payload. Each of the n bacterial strains can produce the same payload. Each payload may be therapeutic. The payload of the mth bacterial strain produces the mth substrate by acting directly or indirectly on the substrate of the (m-1) th bacterial strain, and wherein the payload of the first strain acts on the substrate present in the environment in which the n bacterial strains are cultured. The value of n may be 4. The value of n may be 5. The value of n may be 6. The method may further comprise culturing or co-culturing one or more additional bacterial strains in the culture. Each of the one or more additional bacterial strains may comprise: a lytic plasmid having a lytic gene under the control of an activatable promoter; and an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules; wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule; wherein the quorum sensing molecule in each of the first, second, third and one or more additional bacterial strains may be the same or different, wherein each quorum sensing molecule of each of the first, second, and third bacterial strains and the one or more additional bacterial strains may or may not have any effect on the activatable promoter of the lytic gene of another bacterial strain used in the culture. The lytic plasmid of each of the one or more additional bacterial strains can include a toxin system. The lytic plasmid and the activator plasmid of each of the at least first, second and third strains may be the same plasmid. The lytic plasmid and the activator plasmid of each of the at least first, second and third strains may be independent plasmids. At least the first, second and third bacterial strains are metabolically competitive. At least the first, second and third strains may be selected from escherichia coli, salmonella typhimurium or bacterial variants thereof. Each of the at least first, second and third strains cannot have a growth advantage over the other strain in the culture. In each of at least the first, second and third strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene. The lytic gene in each of the at least first, second and third strains may be E from the bacteriophage Φ X174. The activatable promoter in each of the at least first, second and third bacterial strains may be a LuxR-AHL activatable luxI promoter and the activator gene may be luxI. At least one reporter gene is selected from the group consisting of genes encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP) or variants thereof. The degradation tag may be an ssrA-LAA degradation tag. At least one of the plasmids may be integrated into the genome of at least one of the first, second and third bacterial strains. The culturing can be performed in a specified growth environment. The culturing may be performed in a microfluidic device. The culturing may be performed in a bioreactor. The culturing may be performed in vivo. Each bacterial strain can produce a therapeutic payload and can be cultured in a human or animal patient in need of a therapeutic payload. Each of the first, second, and third time periods may vary from about 12 hours to about 72 hours. Each of the first, second, and third time periods may vary from about 1 hour to about n hours. Each of the first, second, and third time periods may be selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours. Each of the n time periods may partially or completely overlap. The n bacterial strains can be co-cultured. Each of the n bacterial strains was added to the culture in turn, so that each m-th bacterial strain could be added to the culture after passage for a period of (m-1). The first, second and third time periods may be selected from 12 hours, 24 hours, 48 hours, 72 hours and 96 hours.

Also provided herein is a method of maintaining co-culture comprising co-culturing at least three bacterial strains, wherein each of the at least three bacterial strains comprises a lytic plasmid having a lytic gene under the control of an activatable promoter and an activator plasmid having an activator gene, the expression of which promotes accumulation of a quorum sensing molecule, wherein both the activatable promoter of the lytic gene and the expression of the activator gene are activatable by the quorum sensing molecule, wherein the quorum sensing molecule may be different in each of the at least three bacterial strains, wherein each quorum sensing molecule of each of the at least three bacterial strains has no substantial effect on the activatable promoter of the lytic gene of another bacterial strain, wherein the lytic plasmid comprises a toxin/antitoxin system.

Some embodiments of the methods described herein can have one or more of the following features. The toxin/antitoxin system may produce a toxin/antitoxin pair and a different antitoxin of another strain. The lytic plasmid and the activator plasmid of each of the at least three bacterial strains may be the same plasmid. The lytic and activator plasmids of each of the at least three bacterial strains may be isolated plasmids. At least these three strains are metabolically competitive. Each of the at least three bacterial strains may be selected from escherichia coli, salmonella typhimurium, or a bacterial variant thereof. Each of the at least three strains has no growth advantage over the other strain. In each of the at least three strains, the lytic plasmid may comprise a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid may comprise an activator gene, an optional degradation tag, and an optional reporter gene. The lytic gene of at least three strains may be the E of Φ X174 phage. The activatable promoter may be a LuxR-AHL activatable luxI promoter and the activator gene may be LuxI. At least one reporter gene is selected from the group consisting of genes encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP) or variants thereof. The degradation tag may be an ssrA-LAA degradation tag. The co-culture can be inoculated at a ratio of 1:1:1 for each of at least three bacterial strains. At least one of the plasmids may be integrated into the genome of at least one of the at least three bacterial strains. The culturing may be performed in a microfluidic device. The time period may be 12-72 hours. The time period may be selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours. The time period may be selected from 12 hours, 24 hours, 48 hours, 72 hours and 96 hours. Co-cultivation of at least three bacterial strains may be in a constant lytic state; wherein the constant lytic state is characterized by a steady state balance of growth and lysis of at least three bacterial strains.

The methods, systems, kits, bacterial strains, and compositions described herein provide various advantages. First, a triple or multiple lysis system can provide specific delivery of a therapeutic protein via an engineered strain over a period of time until a subsequent strain in the system is administered, thereby spatially and temporally segregating the delivery of the specific therapeutic protein as desired. Otherwise this ability is not available in engineered bacterial treatment methods because they colonize the disease site for a long time, possibly producing a single therapeutic effect regardless of whether another strain enters the disease environment, and at the same time, the colonisation ability of future strains is reduced due to the reduced resources in the disease environment caused by the previously introduced strain.

Second, triple or multiple cleavage systems can also be designed to export a specific pattern of therapeutic proteins to the disease site. For example, the proteins produced by these three strains and the time for their colonization at the disease site may be adjusted depending on the application.

Third, a triple or multiple lysis system can ensure the stability of the loop components in the strain. This can be achieved because subsequent strains in the three lytic cycles are able to kill and remove previous strains at the site of disease, even if members of the population have mutated the loop mechanism. For example, strain a is administered and allowed to colonize the disease site for a desired period of time, wherein members of the population have mutated lytic genes (or any other genes critical to circuit function). Strain B is then introduced into the disease site, allowing the population of strain a to be removed from the environment, including mutant members of the population, because they are not immune to the toxin released by strain B.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials used in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

FIG. 1 shows a strategy for rapid and reliable control of active populations in continuous culture. FIG. 1A is a schematic of a two strain system, wherein strain A has an engineering selectivity advantage over strain B using a geminal toxin-antitoxin system. Strain a produces toxin a, antitoxin a and antitoxin B, while strain B produces only toxin B and antitoxin B. FIG. 1B shows exemplary growth of strain B in FIG. 1A in a microfluidic cell chamber. After introduction of strain a by intermittent loading (grey shaded area), the number of parts of strain a increases exponentially, while the number of parts of strain B decays. The lower panel shows the phase difference and the composition of fluorescence at the indicated time points. AU, arbitrary fluorescence units (background subtracted). FIG. 1C is an exemplary genetic map of a toxin-antitoxin system integrated with a quorum sensing synchronous lytic loop. The circuit contains a lytic plasmid containing a toxin, an antitoxin system, and an activator/reporter plasmid. Transient production of LuxI ultimately leads to accumulation of AHL above the population threshold required to activate LuxR, which initiates a positive feedback loop by driving transcription of the PluxI promoter, which controls production of LuxI, reporter fluorescent protein (sfGFP, CFP or mKAte2) and lytic gene X174E, off. In this system, the LuxR is driven by the native LuxR promoter. FIG. 1D is a schematic of exemplary dual strain population dynamics incorporating a synchronized lysis loop to control population size. The self-limiting behavior of the simultaneous lysis loop, and the simultaneous toxin release when the original strain is in its lowest population, should enable faster strain takeover. FIG. 1E shows a film strip showing the phase contrast, GFP and CFP fluorescence generated by the oscillation circuit. Integration of the simultaneous lysis loop (bottom bar) reduces the time required to achieve complete take-over of the microfluidic trap compared to the non-simultaneous lysis loop strain (top bar). Note: sfGFP and CFP fluorescent reporters of the synchronized lytic strains were driven by AHL production and were only expressed when the population threshold was reached.

Figure 2 shows an exemplary diagram of a three strain system with unidirectional cycling between engineered strains. FIG. 2A is an exemplary schematic of a three strain system for autolytic E.coli. Each strain produces its own toxin-antitoxin pair and also produces the antitoxin of subsequent strains. All three strains utilized the same Lux-AHL quorum sensing system to drive expression of fluorescent reporter protein, as well as self-limiting synchronous lysis. FIG. 2B shows an exemplary batch culture growth curve for 3 strains, showing the lytic OD600 of each strain. All strains started at the same dilution density and were all under the same growth conditions (n-3). Figure 2C shows an exemplary lysis cycle (n-15) for a three strain system measured in a two-layer microfluidic chamber under the same growth conditions. Figure 2D shows co-cultivation of two strains inoculated in a microfluidic device at an initial concentration of 20% of the dominant strain and 80% of the susceptible strain. The strain takes over in one direction, so that the dominant strain takes over the microfluid chamber. Figure 2E shows a video screenshot of co-cultures inoculated with 40% strain 1 and 60% strain 2 at an initial density, showing strain takeover of strain 1 in a double-layer microfluidic chamber. Figure 2F shows a video screenshot of co-cultures inoculated with an initial density of 40% strain 2 and 60% strain 3, showing strain takeover of strain 2 in a double-layer microfluidic chamber. Figure 2G shows a video screenshot of co-cultures inoculated at an initial density of 40% strain 3 and 60% strain 1, showing strain takeover of strain 3 in a double-layer microfluidic chamber. FIGS. 2H, 2I, and 2J each show a time series of fluorescence expression for the microfluidic experiments shown in FIGS. E-G, showing simultaneous lysis and strain takeover of two strains in the microfluidic chamber. The fluorescent reporter of the synchronized lytic strain is driven by AHL production and is only expressed when the population threshold is reached.

FIG. 3 shows the kinetics, model and experimental results of the co-cultivation of three strains. FIG. 3A shows the time series kinetics of the colicin model, indicating that when three strains were co-cultured simultaneously and under the estimated parameters, the three strains should each be cycled once until a single strain takes over the trap. The three panels show the concentration of the strain, colicin and AHL as a function of time. Fig. 3B shows the time traces of GFP, CFP and RFP fluorescence expression for a single well in a 3 strain co-culture experiment, in which all three strains were loaded at approximately equal rates. Fig. 3C shows a video shot of phase difference, GFP, CFP and RFP of the microfluidic chamber shown in fig. 3B. Fig. 3D shows the time traces of GFP, CFP and RFP fluorescence expression of a single well in the experiment of fig. 3C, showing cycling through all 3 strains in a single continuous uninterrupted run. FIG. 3D shows the time series kinetics of the colicin model, indicating that three strains should circulate indefinitely at a given constant inoculum size when they are co-cultured simultaneously under parameter estimation. FIG. 3E shows the limiting cycle of the three strain system, showing a stable transition between the three strains. Furthermore, all reasonable values of the input parameters will result in convergence to the stability limit cycle. Figure 3F shows a graph representing strain takeover events observed in 1600 microfluidic wells, in which all three engineered strains were loaded simultaneously at approximately equal ratios. Arrows indicate each possible transition event between the three strains, and the arrow color indicates the number of transitions observed for each arrow.

Fig. 4 illustrates exemplary toxins that may be used according to some embodiments.

FIG. 5 shows an exemplary validation of colicin and immune activity in a non-lytic strain in a 96-well plate reader assay. FIG. 5A shows a MG1655 wild-type control strain with an mKate2 expression plasmid. Time course traces of OD600 and RFP fluorescence expression. Fig. 5B shows a MG1655 wild-type control strain with a GFP expression plasmid. Time course traces of OD600 and GFP fluorescence expression (RFU). FIG. 5C shows the time course traces of OD600 of MG1655 strain 1(pML0001) and GFP fluorescence expression. FIG. 5D shows the time course traces of OD600 and RFP fluorescence expression of MG1655 strain 2. FIG. 5E shows the time course traces of OD600 and RFP fluorescence expression of MG1655 strain 3. FIG. 5F shows co-cultivation of MG1655 strain 1 with colicin E3. The time course traces of OD600, GFP and RFP Fluorescence (RFU) expression confirmed that strain 1 was able to grow in the presence of colicin E3. FIG. 5G shows the co-incubation of MG1655 strain 2 with colicin E7. The time course traces of OD600 and GFP fluorescence (RFU) expression confirm that Strain 1 can grow in the presence of colicin E7. FIG. 5H shows the co-incubation of MG1655 strain 3(pML0004) with colicin V. The time course traces of OD600 and RFP fluorescence expression confirmed that strain 3 was able to grow in the presence of colicin V. FIGS. 5I, 5J and 5K show the effectiveness of colicin to inhibit the growth of wild-type MG1655 as demonstrated by cocultivation of colicin E3, E7 and V with a wild-type MG1655 control strain. Time course traces of OD600, GFP and RFP fluorescence (AU).

Figure 6 shows video screenshots taken from an exemplary microfluidic experiment showing that growth of one strain is inhibited by toxin production of another strain. In this experiment, strain 1 produced toxin 1, antitoxin 1 and antitoxin 3. Strain 3 produced toxin 3, antitoxin 3, and antitoxin 2. When comparing the normal growth of strain 3 (scheme 2) with the growth rate of strain 1 when in the vicinity (scheme 3), the growth inhibitory effect of the toxin released by strain 1 was evident. The results show that toxin activity is proximity dependent, as shown (scheme 4) when the two strains are not in proximity, the growth of strain 3 is not affected. Figure 6A is scheme 1 showing the individual growth of strain 1 in a microfluidic well. Figure 6B is scheme 2 showing the growth of strain 3 alone in a microfluidic well. Fig. 6C is scheme 3, showing growth and strains 1 and 3 when strains 1 and 3 are close to each other. Strain 1 inhibited the growth of Strain 3. FIG. 6D is scheme 4 showing growth of strains 1 and 3 when not in proximity to each other. FIGS. 6E and 6F show fluorescence traces for different protocols in GFP and RFP.

FIG. 7 shows typical characteristics of the disrupted strain in plate readers and microfluidic experiments. The amplitude of transmitted light oscillations in microfluidic experiments varies due to differences in the phenotype of the cleavage event. For example, the lysis event of strain 1 will occur more vigorously, driving cell lysate out of the trap. However, the lysis event of strain 3 resulted in the accumulation of more cell lysate in the microfluidic well. FIG. 7A shows the plate reader OD lysed for 3 lytic strains ₆₀₀And the corresponding fluorescent expression (n-3). Fig. 7B shows a superposition of the fluorescence intensity normalized to the maximum value (solid line) and the transmitted light intensity normalized to the maximum value (dashed line) for strain 1 of the microfluidic well region (100x80x2 μm). Fig. 7C shows a superposition of the fluorescence intensity normalized to maximum (solid line) and the transmitted light intensity normalized to maximum (dashed line) for strain 2 of the microfluidic well region (100x80x2 μm). Fig. 7D shows a superposition of the fluorescence intensity normalized to maximum (solid line) and the transmitted light intensity normalized to maximum (dashed line) for strain 3 of the microfluidic well region (100x80x2 μm).

Figure 8 shows the deterministic modeling of an exemplary three strain three lysis system. FIG. 8A shows as A_cThe magnitude of the non-zero eigenvalues of the plotted mode of the function. When λ 23 is 1, in A_cThe transition to oscillation occurs at 1.25. Fig. 8B shows the number of cleavage events per switching cycle in the vicinity of the Hopf bifurcation. Fig. 8C shows the Hopf bifurcation plot of the mapping model. Fig. 8D, 8E, 8F and 8G each show a parameter sweep evaluating the dependency of the cleavage event interval and the number of cleavage events per switching cycle on the system parameters in the oscillatory regime.

Figure 9 shows approximately 1600 individual exemplary microfluidic wells from 4 independent 3 strain co-culture experiments analyzed. The microfluidic device was loaded with all 3 engineered strains simultaneously, thereby varying the initial seeded cell concentration per microfluidic well. The traps demonstrating the transition between strains are recorded and shown in the upper graph. The colors (grey, black, light grey) correspond to strain 1, strain 2 and strain 3, respectively, representing the dominant strains in the wells at a given experimental time. Fig. 9A shows the transition event from strain 1 (grey) to strain 3 (light grey) over time. Fig. 9B shows the transition event from strain 2 (black) to strain 1 (grey) over time. Fig. 9C shows the transition event from strain 3 (light grey) to strain 2 (black) over time. Fig. 9D shows the transition events from strain 1 (grey) to strain 3 (light grey) to strain 2 (black) over time. Fig. 9E shows the transition event from strain 2 (black) to strain 1 (grey) to strain 3 (light grey) over time.

FIG. 10 shows exemplary strains and gene expression constructs found on each plasmid that can be used according to some embodiments.

FIG. 11 shows exemplary plasmids that can be used according to some embodiments.

Figure 12 shows an exemplary extension of the function of the plasmid-stabilized toxin-antitoxin module in a multi-strain population. Figure 12A shows that in a monoclonal population, the plasmid-stable toxin-antitoxin module is able to kill progeny cells that have mutated or lost the antitoxin gene. When extended to a system of n >1 strains, the strain containing the TA module maintained self-stabilizing function while gaining additional ability to kill any strains without antitoxin. Fig. 12B is a schematic showing that each strain produces its own toxin-antitoxin pair while also producing an antitoxin of a subsequent strain, thereby achieving sequential strain inhibition. Strain R produces toxin 1, antitoxin 1 and antitoxin 2. Strain P produces toxin 3, antitoxin 3 and antitoxin 1. Strain S produces toxin 2, antitoxin 2 and antitoxin 3. FIG. 12C is a universal plasmid map of RPS strains. Fig. 12D is a plot of batch culture growth rate of engineered RPS and wild-type e.coli MG1655 strain (n-3). All strains were started from the same dilution density and under the same growth conditions. Fig. 12E is a set of histograms depicting approximate population share over time for strains co-cultures inoculated at an initial ratio of 1:2 dominance to susceptibility (n-8). From top to bottom: (1) strain R (light grey) inhibits strain S (black); (2) strain S (black) inhibits strain P (grey); (3) strain P (grey) inhibits strain R (light grey). Fig. 12F is a fluorescence microscope image showing the composition of phase contrast, GFP, CFP and RFP fluorescence. From left to right: (1) strain R (light grey) inhibits strain S (dark grey); (2) strain S (dark grey) inhibits strain P (grey); (3) strain P (grey) inhibits strain R (light grey).

Fig. 13 shows an exemplary development of an engineered stone-scissors-cloth ecology. Figure 13A is a schematic of exemplary dual strain population dynamics with SLC integration. Continuous synchronized population splitting during strain takeover events is described. FIG. 13B is an exemplary genetic map of a quorum-sensing synchronous resolution loop and TA module. The first plasmid contained the X174E cleavage protein and the corresponding toxin/antitoxin gene driven by the luxI promoter. The second "activator" plasmid contains the luxI and LuxR genes driven by its native promoter, plluxi. Fig. 13C is a schematic showing that each strain produced its own toxin-antitoxin pair, while also producing antitoxins of subsequent strains. All three strains utilized the same Lux-AHL quorum sensing system to drive expression of fluorescent reporter protein, as well as self-limiting synchronous lysis. Fig. 13D shows the time series of fluorescence expression and video screenshots of co-cultures inoculated with strain 1 and strain 2 at a 1:1 ratio, showing strain takeover of strain 1 in the microfluidic chamber. Fig. 13E shows the time series of fluorescence expression and video screenshots of co-cultures inoculated with strain 2 and strain 3 at a 1:1 ratio, showing strain takeover of strain 2 in the microfluidic chamber. Fig. 13F shows the time series of fluorescence expression and video screenshots of co-cultures inoculated with strain 3 and strain 1 at a 1:1 ratio, showing strain takeover of strain 3 in the microfluidic chamber. Fig. 13G is a graph illustrating the limit cycle of the three-strain system, showing convergence to a stable transition between the three strains regardless of the initial strain ratio. FIG. 13H is a graph showing strain takeover events observed when all three engineered strains were cultured simultaneously. Arrows indicate each possible transition event between the three strains, and arrow shading indicates the number of transitions observed for each arrow.

Figure 14 shows an exemplary extension of circuit function in the absence of antibiotics. FIG. 14A is a schematic of scheme 1, depicting a system where oscillations between the growth phase and the lytic transition become uncontrolled growth due to loss of circuit function. Scheme 2 describes a cycle consisting of a three strain system in which the transition to uncontrolled growth is prevented by the addition of the next strain of the system. Fig. 14B is a time trace of fluorescent expression for each strain, showing loss of loop function over time. According to scheme 1 in FIG. 14A, each strain was cultured for 32 hours in the absence of kanamycin. Fig. 14C is a boxed graph (n ═ 16) depicting the time to loss of function of each strain. Fig. 14D: according to scheme 2 in fig. 14A, the strains were loaded sequentially, starting from strain 2 and strain 1. Top: time traces of GFP, CFP and RFP fluorescence expression of a single well, showing cycling through all 3 strains in a single continuous uninterrupted run. Bottom-video shot of phase difference and fluorescence composition at specified time points. AU, arbitrary fluorescence units (background subtracted).

Figure 15 shows exemplary effects of RPS on mutable genetic circuits. Fig. 15A shows scheme 1, which depicts a system in which strain 1 is cultured in 96-well plates for the duration of a single lysis event and passaged every 12 hours. Scheme 2 describes a system in which strain 1 is cultured in 96-well plates for a period of a single lysis event and passaged every 12 hours. At every third passage, the next strain of the RPS system was added to the culture at the same time. Fig. 15B shows a side-by-side comparison of each protocol in fig. 15A (n-12) over the duration of 12 passages. Light grey squares represent functionally synchronized population lysis. Grey squares represent loss of synchronized population lysis. Dark grey squares represent the loss of simultaneous population lysis for subsequent recovery. In addition, 12 corresponding samples in each protocol were Sanger sequenced in the 1000 base pair region containing the X174E cleavage protein and the Lux cassette. The check mark indicates a correct sequence, (X) indicates an incorrect sequence, and (i) indicates an indeterminate sequence read. All strains started from the same dilution density and were all under the same growth conditions.

FIG. 16 shows an exemplary validation of colicin activity in a non-lytic strain in a 96-well plate reader assay. FIG. 16A shows MG1655 wild-type E.coli OD₆₀₀Time course trajectory. FIG. 16B is MG1655 wild type E.coli OD co-incubated with filtered lysate of colicin V producing strain₆₀₀Time course trajectory. FIG. 16C is MG1655 wild type E.coli OD co-incubated with filtered lysate of colicin E7 producing strain₆₀₀Time course trajectory. FIG. 16D is MG1655 wild type E.coli OD co-incubated with filtered lysate of colicin E3-producing strain₆₀₀Time course trajectory.

FIG. 17 shows typical characteristics of strains in plate readers and microfluidic experiments. FIG. 17A is the plate reader OD of lysis of 3 lysis strains₆₀₀And the corresponding fluorescence expression (n-3). Fig. 17B is a superimposed graph of the fluorescence intensity normalized to the maximum value (solid line) and the transmitted light intensity normalized to the maximum value (dashed line) of strain 1 of the microfluidic well region (100x80x1.2 μm). Fig. 17C is a superimposed graph of the fluorescence intensity normalized to the maximum value (solid line) and the transmitted light intensity normalized to the maximum value (dashed line) of strain 2 of the microfluidic well region (100x80x1.2 μm). Fig. 17D is a superimposed graph of the fluorescence intensity normalized to the maximum value (solid line) and the transmitted light intensity normalized to the maximum value (dashed line) of strain 3 of the microfluidic well region (100x80x1.2 μm).

Fig. 18 shows an exemplary co-culture incubation of pairs of strains in a microfluidic device. Fig. 18A is a histogram showing that each pair of strains was co-cultured at a ratio of dominant (bold) to susceptible (italics) 1:1 (n-35). For each strain pair, the dominant strain replaced the susceptible strain in 100% of the culture area. Fig. 18B is a histogram showing that each pair of strains was co-cultured at a ratio of dominant (bold) to susceptible (italics) 1:5 (n-396). The dominant strain of each pair replaced the susceptible strain in 100%, 100% and 92% of the culture area.

Figure 19 shows typical time series kinetics of the colicin model, assuming the system is shut down and no new cells are introduced. The three panels show the concentration of strain, colicin and AHL over time when the initial concentration of strain 3 was high and no new cells were introduced.

FIG. 20 shows typical three strain co-cultivation kinetics. FIG. 20A is a time trace of GFP, CFP and RFP fluorescence expression of a single well in a 3 strain co-culture experiment in which all three strains were loaded at approximately equal rates. Fig. 20B shows video screenshots of phase difference, GFP, CFP and RFP for the microfluidic chamber shown in fig. 20A.

FIG. 21 shows exemplary plasmids that can be used according to some embodiments.

Fig. 22 illustrates exemplary toxins that may be used according to some embodiments.

Fig. 23 shows exemplary strains that can be used according to some embodiments.

FIG. 24 shows γ_A＝1,γ_C＝0.2,α＝1,β＝1,δ＝0.02,A_cExemplary kinetics of the colicin model of 1, ∈ 0.001. Fig. 24A shows an exemplary time series. FIG. 24B shows P_i,Q_i,R_iExemplary plots of (a).

FIG. 25 shows γ_A＝10,γ_C＝1,α＝10,β＝1,δ＝0.002,A_cExemplary kinetics of the colicin model at 100, 0.001. Fig. 25A shows an exemplary time series. FIG. 25B shows P_i,Q_i,R_iExemplary plots of (a).

FIG. 26 shows γ_A＝10,γ_C＝1,α＝10,β＝20,δ＝0.01,A_cExemplary kinetics of the colicin model 100, 0.02. Fig. 26A shows an exemplary time series. FIG. 26B shows P_i,Q_i,R_iExemplary plots of (a).

Detailed Description

Population control is a powerful tool in synthetic biology. In the past decade, control of microbial communities has been achieved in some systems. Population control can be divided up into two major systems to date. The first is a single strain community, exhibiting self-limiting population density. The mechanism behind these systems utilizes a bacterial quorum sensing system, such as the LuxI/LuxR AHL system. When cells grow to a certain density and reach a "population" (quorum), they produce a protein that is toxic or induces lysis. The second category can be categorized as two strain populations, where the population exhibits predator-prey behavior, oscillatory behavior, or maintains a constant ratio between the two strains. Two strain systems, much like single strain systems, rely on quorum sensing to drive the production of toxins or lytic proteins within each strain. For example, in the predator-prey model, a two-way quorum sensing system is used to drive the production of toxic proteins in prey only when predators are present. Other mechanisms of two strain systems include the use of orthogonal quorum sensing systems, such as Lux and Las, to drive oscillatory lytic events between the two strains, followed by population density oscillations over a period of time.

Advances in synthetic biology have led to a collection of proof-of-principle bacterial circuits that can be utilized in applications ranging from therapeutics to biological production. For most applications, a common challenge is the presence of selective stress that can lead to high mutation rates of engineered bacteria. One common strategy is to develop cloning techniques aimed at increasing the fixation time of deleterious mutations in single cells. Provided herein is a complementary method guided by ecological interactions in which periodic population controls are designed to stabilize the function of intracellular gene circuits.

In the past two decades, synthetic biologists have developed complex Molecular loops to control the activity of various cells (see, e.g., M.B.Elowitz, S.Leibler, Nature 403,335 (2000); T.S.Gardner, C.R.Cantor, J.J.Collins, Nature 403,339 (2000); J.Hasty, D.McMillen, J.J.Collins, Nature 420,224 (2002); L.You, R.S.Cox III, R.Weiss, F.H.Arnold, Nature 428,868 (2004); P.E.Purnick, R.Weiss, Nature reviews Molecular biology 10,410 (2009); H.H.Wang, et al., (460, 894); S.2009).D.M.Müller,M.Wieland,M.Fussenegger, Nature 487,123 (2012); p.siuti, j.yazbek, t.k.lu, Nature biotechnology 31,448 (2013); t.h.segall-Shapiro, e.d.sontag, c.a.voigt, Nature biotechnology 36,352 (2018)). Over time, these systems inevitably become dysfunctional due to evolutionary selection pressure, leading to uncontrolled mutations (see, e.g., f.k.balagade, l.you, c.l.hansen, f.h.arnold, s.r.quake, Science 309, 137 (2005)). Methods to address this challenge include integration of recombinant elements into the host genome (see, e.g., m.y.peredlechuk, g.n.bennett, Gene 187231(1997)), or the use of plasmid stabilizing elements (see, e.g., k.gerdes, Nature Biotechnology 61402(1988)), synthesis of "kill switches" (see, e.g., c.t.chan, j.w.lee, d.e.cameron, c.j.bashor, j.j.collins, Nature chemistry 12, 82(2016)), or synthesis of amino acids (see d.j.mantel et al, Nature 518, 55 (2015); a.j.rovner et al, Nature 518, 89 (2015); n.ostov et al, 353, 2016; j.w.819, c.t.t.c.c. channel, s.2012018, Nature 1 (2016)). Although the stabilizing element may extend toward the mutation, evolution inevitably renders the stabilizing element ineffective (see, e.g., f.k. balagade, l.you, c.l. hansen, f.h. arnold, s.r. quake, Science 309, 137 (2005)). This is particularly true in vivo applications where antibiotics are difficult to deliver (see, e.g., w.c. ruder, t.lu, j.j.collins, Science 3331248 (2011); m.a.fischbach, j.a.bluestone, w.a.lim, Science transactional 5179ps7 (2013); d.t.riglar, p.a.silver, Nature Reviews Microbiology 16214 (2018); m.o.din, et al., Nature 536, 81 (2016); a.p.teixeira, m.fussenger, biotechnology view 47, 59 (2017); b.p.landry, j.j.tabor, Microbiology spectrum 2015 (2017); or where the function of the latest plasmid was interrupted (j.j.j.j.j.t., Science 2015, di), or where the function of the latest plasmid was particularly problematic (j.g.h.h.798, j.h.r.798). Described herein is how to design a small ecosystem to stabilize gene circuit function, thereby complementing genetic engineering at the single cell level. Composition of stability and ecosystem differentiation The same subpopulations are isolated from each other, rather than including stabilizing elements in one strain, such that uncontrolled mutation of one strain does not result in failure of a stable ecosystem.

The field of synthetic biology has evolved over the past decade from single cell engineering to whole population engineering. Described herein are three strain (or more) population control systems and methods that can be cycled continuously over long periods of time, which has not previously been achieved. Now, the triple or multi-lytic system presents a new multi-population dynamic control model (loops in ecology instead of loops in cells) that opens up exciting new possibilities for engineering synthetic systems. These multi-strain systems allow scientists to achieve complex interactions that were previously not possible through engineering of single cells or single populations.

Disclosed herein are methods, materials, and circuits/devices/systems involving multi-strain "triple lysis" or "multi-lysis" microbial systems. A "scissors stone cloth" system provides a unique solution to the synthetic biology problem of plasmid loss or mutation in a complex circuit due to selection pressure. For example, three strains of E.coli can be designed such that each strain can kill the other two strains, or one of the other two strains. The resulting "stone scissors cloth" ecology showed rapid cycling of the strains in the microfluidic device and resulted in a significant increase in the stability of gene circuit function in cell culture. This can be achieved by a triple or multi lysis system, since subsequent strains in a triple or multi lysis cycle are able to kill and remove the previous population of strains, even if members of the population have mutated the loop mechanism. The "triple lysis" or "multiple lysis" systems use at least three bacterial strains to kill each strain complementarily, either periodically or in a specific time manner. For example, strain a is administered and allowed to colonize a region, over a period of time, the population may begin to mutate lytic genes (or any other genes critical to circuit function). As another example, strain a is administered and colonized in an area, over a period of time, the population may develop mutations in a gene of interest (e.g., a gene encoding a heterologous nucleic acid and/or protein, e.g., a therapeutic agent or payload). Strain B is then introduced into the ecosystem, allowing the population of strain a to be removed from the environment, including mutant members of the population, since they are not immune to the toxins released by strain B. The use of a system as provided herein as a method of ensuring circuit stability has many broad applications in the fields of therapeutic, production and sensing applications. As used herein, "lysis" includes the process of breaking down cells in a manner that compromises the integrity of the cells, as well as any process that kills the cells. Exemplary processes of cell death encompassed by "lysis" include expression of lytic genes, action of toxins, and the like. In some exemplary embodiments, toxin-induced cell death may occur through a process that breaks down cells in a manner that compromises their integrity. In some embodiments, the toxin can trigger cell death without decomposing the cell.

Any of the toxin systems described herein may be replaced by an appropriate cell death system. In some cases, the cell death system may include different pairs of substances (e.g., toxin-like antitoxins). In other cases, different characteristics of adjacent or consecutive bacterial strains may be specific for a previous strain (e.g., a strain may produce substances that are specific and harmful to a previous strain, but which are not themselves damaged by such substances due to certain different characteristics compared to a previous strain). Other exemplary pairs of substances that may be used herein are as follows: (i) antibiotic or antibacterial peptides and host defense peptides (e.g., obtained from bacterial, fungal, or animal sources); (ii) a system comprising a bacteriophage specific to a previous strain earlier in the cycle, e.g., the bacteriophage is delivered sequentially by the bacterium to clear the previous strain to which the bacteriophage is specific; (iii) a system comprising a delivery prodrug that kills a previous strain when activated by a bacterial protease specific for the previous strain; (iv) systems comprising the use of different strains in the cycle, wherein each strain is a system that produces the same single toxin as the previous strain in the cycle (e.g., acinetobacter (predator) and escherichia coli (prey); (v) each strain produces the same single toxin, but with orthogonal quorum sensing to activate toxin expression of the previous strain (e.g., an orthogonal quorum system in which production of such toxin in the previous strain is activated by the presence of another strain (via a quorum sensing molecule of the other strain), e.g., to initiate toxin or split protein production in the n-1 strain when the nth strain is added.) furthermore, in some embodiments, the toxin system may also be a toxin (or other type of cell death system, as described herein) specific to some bacterial species but not other strains in addition to the expressed antitoxin. In addition to expressing an antitoxin, a bacterium that expresses a toxin (or other type of cell death system, as described herein) may have properties that make it insensitive to the toxin (or other type of cell death system, as described herein) that it produces that is specific for other strains. For example, different bacterial species may be used, which may be targets for toxins specific to different species (e.g., escherichia coli followed by salmonella) where salmonella produces antimicrobial peptides specific for escherichia coli (e.g., colicin only kills escherichia coli and not salmonella). In such systems, an analog of an antitoxin is inherently insensitive to the toxin that it expresses by subsequent strains, which toxin is specific to the previous strain species. Similarly, other characteristics of the bacterial strains may constitute suitable cell death systems, for example where one strain is a gram-positive bacterium followed by a gram-negative bacterial strain that produces a toxin specific for the gram-positive bacterium (or other type of cell death system, such as the cell death system described herein), with an analog of a gram-negative antitoxin being in a state where it is not a gram-positive bacterium. In other instances, the analog of the bacterial strain antitoxin may be the presence or absence of certain surface receptors or cellular import/export systems as compared to other bacterial strains, e.g., an E.coli strain may have receptors necessary for toxicity of the toxin (or other type of cell death system, as described herein), expressed by an E.coli strain lacking such receptors, e.g., an E.coli strain expressing the SdaC/DcrA receptor (which is necessary for colicin V toxicity), followed by an E.coli strain that does not express the SdaC/DcrA receptor but expresses the colicin V (in which case, in a second strain, the SdaC/DcrA receptor will be similar to the antitoxin Any suitable combination of a toxin system and a cell death system.

In addition, the triple or multiple lysis system also ensures that a specific delivery of a therapeutic protein is provided via the engineered strain over a period of time until the subsequent strain in the system is administered, thereby spatially and temporally isolating the delivery of the specific therapeutic protein. Otherwise this ability is not available in engineered bacterial treatment methods because they colonize the disease site for a long time, possibly producing a single therapeutic effect regardless of whether another strain enters the disease environment, and at the same time, the colonisation ability of future strains is reduced due to the reduced resources in the disease environment caused by the previously introduced strain.

As used herein, the term "co-culture" or "co-culture" refers to growing or culturing two or more (e.g., three or more) different cell types (e.g., at least two different bacterial strains or at least three bacterial strains) within a single recipient or environment (e.g., a single cell culture vessel, a single cell culture plate, a single bioreactor, a single microfluidic device, or a single object (e.g., in vivo)). In certain instances, co-culturing can include growing or culturing two or more (e.g., three or more) different cell types simultaneously. In some cases, co-culturing can include growing or culturing two or more (e.g., three or more) different cell types (e.g., growing a first cell type, followed by a second cell type, followed by a third cell type, where the growth phases can overlap or can be non-overlapping) within a single recipient over a period of time. In some embodiments, co-culturing can include growing or culturing two or more (e.g., three or more) different cell types within a single recipient over a period of time, wherein the growth phases of each different cell type partially overlap.

Any of the plasmids described herein can be introduced into a bacterial cell (e.g., a gram-negative bacterial cell, a gram-positive bacterial cell) using a number of different methods known in the art. Non-limiting examples of methods for introducing nucleic acids into cells include: transformation, microinjection, electroporation, cell extrusion, sonoporation. It will be understood by those skilled in the art that the plasmids described herein may be introduced into any of the cells provided herein.

The term "treating" is used herein to mean delaying the onset of a disease, inhibiting a disease, ameliorating the effects of a disease, or extending the lifespan of a subject having a disease, such as cancer, an infection.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount or concentration of at least three bacterial strains effective to produce the desired effect or physiological result after administration thereof, after use of a composition or treatment described herein for a period of time (including short or long term administration and regular or continuous administration). For example, effective amounts of at least three bacterial strains that express and/or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein) for use in the present invention include, for example: an amount that inhibits the growth of a cancer (e.g., tumor cells and/or tumor-associated immune cells), an amount that ameliorates or delays the growth of a tumor, an amount that improves survival of a subject having or at risk of developing a cancer, and an amount that improves the outcome of other cancer treatments. As another example, an effective amount of at least three bacterial strains that express and/or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein) can include an amount that beneficially affects the tumor microenvironment.

Throughout the specification, the term "subject" (and "patient" are used interchangeably) to describe an animal, human or non-human, to which treatment according to the methods of the invention is provided. The present invention clearly predicts veterinary use. The term includes, but is not limited to, birds, reptiles, amphibians, and mammals such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep, and goats. Preferred subjects are humans, domestic animals and domestic pets such as cats and dogs. In some embodiments, the subject is a human. For example, in any of the methods described herein, the subject may be at least 2 years old or older (e.g., 4 years old or older, 6 years old or older, 10 years old or older, 13 years old or older, 16 years old or older, 18 years old or older, 21 years old or older, 25 years old or older, 30 years old or older, 35 years old or older, 40 years old or older, 45 years old or older, 50 years old or older, 60 years old or older, 65 years old or older, 70 years old or older, 75 years old or older, 80 years old or older, 85 years old or older, 90 years old or older, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16,18, 20, 21, 24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 80, 90, 95, 96, 95, 99, 100, or 100 years old).

The term "population" when used before a noun refers to two or more of that particular noun. For example, the phrase "population of bacterial strains" refers to three or more bacterial strains.

The term "cancer" refers to cells with an unlimited ability to grow autonomously. Examples of such cells include cells having an abnormal state or condition characterized by rapid proliferative cell growth. The term is intended to include cancerous growths, such as tumors, oncogenic processes, metastatic tissues, malignantly transformed cells.

Metastatic tumors can arise from a variety of primary tumor species, including but not limited to tumors of prostate, colon, lung, breast, bone and liver origin. Metastases develop, for example, when tumor cells slough, detach or migrate from the primary tumor, they enter the vascular system, infiltrate into the surrounding tissue, and grow to form a tumor at various anatomical sites, for example, sites distant from the primary tumor.

Individuals identified as at risk for developing cancer may benefit from the present invention, for example, because prophylactic treatment may be initiated prior to the occurrence of any evidence and/or diagnosis of the condition. An "at risk" individual, for example, includes an individual exposed to a carcinogen, e.g., by consumption (e.g., by inhalation and/or digestion), the level of which has been statistically demonstrated to promote cancer in a susceptible individual. Also included are individuals at risk for exposure to ultraviolet radiation, or for their environment, occupation, and/or genetics, as well as individuals who exhibit signs of precancerous conditions such as polyps. Similarly, individuals who are at a very early stage of cancer or metastatic progression (i.e., only one or a few abnormal cells are present in the individual's body or at a particular site in the individual's tissue) may benefit from such prophylactic treatment.

It will be understood by those skilled in the art that a subject may be diagnosed as having or at risk of a condition described herein (e.g., cancer), for example, by a medical professional such as a doctor or nurse (or veterinarian, as appropriate for the subject to be diagnosed) using any method known in the art, for example, by assessing the subject's medical history, conducting diagnostic tests, and/or applying imaging techniques.

It will also be understood by those skilled in the art that the subject need not be treated by the same individual who diagnosed the subject (or who prescribed treatment for the subject). Treatment may be administered (and/or may be administered supervised) by, for example, the individual making the diagnosis and/or prescription, and/or any other individual including the subject himself (e.g., where the subject is capable of self-administration).

Also provided herein are methods of producing a recombinant bacterial cell capable of expressing and/or secreting a therapeutic agent (e.g., any of the therapeutic agents described herein), comprising: a nucleic acid encoding a therapeutic agent produced in a recombinant bacterial cell, and optionally a plasmid stabilizing element, wherein the plasmid stabilizing element is a toxin/antitoxin system; and culturing the recombinant bacterial cell under conditions sufficient for expression and/or secretion of the toxin, antitoxin, and therapeutic agent. Also provided herein are methods of producing a recombinant bacterial cell capable of expressing and/or secreting a therapeutic agent (e.g., any of the therapeutic agents described herein), comprising: introducing into a bacterial cell a lytic plasmid, an activator plasmid, a nucleic acid encoding a therapeutic agent to be produced in the recombinant bacterial cell, and a plasmid stabilizing element; and culturing the recombinant bacterial cell under conditions sufficient for expression and/or secretion of the toxin, antitoxin, and therapeutic agent. In some embodiments, the plasmid stabilizing element is a toxin/antitoxin system. In some embodiments, the plasmid stabilizing element is expressed in a lytic plasmid. In some embodiments, the toxin/antitoxin system can produce a toxin/antitoxin pair and a different antitoxin of another strain. In some embodiments, the introducing step can comprise introducing into the recombinant bacterial cell an expression vector comprising a nucleic acid encoding the therapeutic agent to be produced in the recombinant bacterial cell. In some embodiments, the bacterial cell is an escherichia coli cell, a salmonella typhimurium cell, or a bacterial variant thereof. In some embodiments, the bacterial strain is a gram-negative bacterial strain, such as a Salmonella (Salmonella), Acetobacter (Acetobacter), Enterobacter (Enterobacter), clostridium (Fusobacterium), Helicobacter (Helicobacter), Klebsiella (Klebsiella), or escherichia coli (e. In some embodiments, the bacterial strain is a gram-positive bacterial strain, such as an actinomycete (actinomycete) strain, a Bacillus (Bacillus) strain, a Clostridium (Clostridium) strain, an Enterococcus (Enterococcus) strain, or a Lactobacillus (Lactobacillus) strain. In some embodiments, the at least three bacterial strains are all gram-negative bacterial strains or all gram-positive bacterial strains. In some embodiments, at least one of the at least three bacterial strains is a gram-negative bacterial strain. In some embodiments, at least one of the at least three bacterial strains is a gram-positive bacterial strain.

Methods of culturing bacterial cells are well known in the art, and examples of such methods are provided in the examples. Bacterial cells can be maintained in vitro under conditions conducive to proliferation and growth. Briefly, bacterial cells can be cultured by contacting bacterial cells (e.g., any of the bacterial cells described herein) with a cell culture medium that includes the necessary growth factors and supplements that support cell viability and growth.

Methods for introducing nucleic acids and expression vectors into bacterial cells are known in the art. For example, transformation can be used to introduce nucleic acids into bacterial cells. In some embodiments, the bacterial cell can comprise any of the plasmids described herein.

Provided herein is a bacterial strain comprising a toxin system (also referred to as a toxin/antitoxin system) comprising a toxin/antitoxin pair (e.g., an antitoxin is effective against the toxin). In some embodiments, the toxin/antitoxin system can produce a toxin/antitoxin pair and a different antitoxin of another strain. The toxin system may be included (e.g., encoded) in any suitable location. In some embodiments, the toxin system may be contained in the genome of the bacterial strain. In some embodiments, the toxin system may be contained in one or more plasmids. In some embodiments, the toxin system may be partially contained in the genome and partially contained in one or more plasmids. In some embodiments, the toxin system may be comprised in a lytic plasmid. In some embodiments, the toxin system can be contained in an activator plasmid. In some embodiments, the toxin system can be contained in a third plasmid. The third plasmid is any suitable plasmid. In some embodiments, the toxin system may be engineered or naturally occurring in the bacterial strain. In some embodiments, the toxin system may be inserted into a bacterial strain.

The toxin system may be any suitable toxin system. In some embodiments, the toxin system comprises a first toxin/first antitoxin pair (e.g., a first antitoxin effective against a first toxin) and a second antitoxin. In some embodiments, the strain encoding such a toxin system does not encode a second toxin against which a second antitoxin is effective.

In some embodiments, the toxin/antitoxin system can be a type I toxin/antitoxin system, a type II toxin/antitoxin system, a type IV toxin/antitoxin system, a type V toxin/antitoxin system, or a type VI toxin/antitoxin system. Non-limiting examples of type I toxins/antitoxins include Hok and Sok, Fst and RNAII, TisB and IstR, LdrB and RdlD, FlmA and FlmB, Ibs and Sib, TxpA/BrnT and RatA, SymE and SymR, and XXCV2162 and ptaRNA 1. Non-limiting examples of type II toxins/antitoxins include CcdB and CcdA; ParE and ParD; MaxF and MazE; yafO and yafN; HicA and HicB; kid and Kis; zeta and Epsilon; DarT and DarG. For example, the type III toxin/antitoxin system involves an interaction between a toxic protein and an RNA antitoxin, such as ToxN and ToxI. For example, the type IV toxin/antitoxin system includes a toxin/antitoxin system that counteracts the activity of the toxin and that the two proteins do not directly interact with each other. An example of a type V toxin/antitoxin system is GoT and GoS. An example of a type VI toxin/antitoxin system is SocA and SocB.

In some embodiments, the toxin/antitoxin system may be replaced by a bacteriocin/immune protein system. As used herein, the term "toxin/antitoxin" includes the toxin/antitoxin system described above, including the bacteriocin/immune protein system. Bacteriocins are ribosomally synthesized peptides produced by bacteria. Bacteriocins are not toxic to the bacteria producing the bacteriocins, but are generally toxic to other bacteria. Typically, the bacteriocin-producing bacteria also produce an immunity protein that inhibits or prevents the toxic effects of the bacteriocin. Thus, bacteriocins and corresponding immunity proteins can be used in a manner similar to the toxin/antitoxin systems described herein. Most bacteriocins are very potent and exhibit antimicrobial activity at nanomolecular concentrations. For example, a eukaryote-producing microorganism has 10²To 10³Lower activity (Kaur and Kaur (2015) front. Pharmacol. doi:10.3389/fphar. 2015.00272).

Non-limiting examples of bacteriocins that can be included in any of the bacterial strains, systems, and methods described herein include: acidophilic lactocin (acidicin), actagardine (actagardine), agrobacterin (agrocin), alveolus haustorins (alveicin), aureomycin (aureocin), aureomycin A53(aureocin A53), aureomycin A70(aureocin A70), bifidin (bisin), leuconostin (carnocin), cyclobacteriocin (carnocyclin), caseicin (caseicin), ceresin (cerein), circulin A (circulan A), colicin (colicin), curvulysin (curvulysin), botulinum kubamicin (digerin), duramycin (duramycin), enterococcin (enterocin), enterolysin (enterolysin), epidermidin (epididymin/gallicin), victoriin (virucin), lactein (cerin), lactein (lactein), garicin (secacin), garicin(s), sorangin (soricin A), soricin (soricin), soricin(s), soricin (soricin A), soricin (soricin), soricin(s), soricin (soricin), soricin A), soricin (soricin), nisin, icilin (leucosin), lysostaphin (lysostaphin), macystin (macedocin), mersacidin (mersacidin), leuconostoc mesenteroides (mestericin), microcystin (micropsporin), microcins S (microcin S), mutans (mutacin), nisin (nisin), paenibacillin (paenibacillin), plankton (planosporin), pediocin (pediocin), pentostatin (pentostatin), phytolaccolicin (plantaricin), pneumococcocin (pneumocciin), pustusin (pycin), reuterin 6 (reuterin 6), qing lactin (kasacin), lisicin (livaccin), bacilin (subtilisin), anti-subtilisin (subtilisin), staphylosin (staphyloccin), staphyloccin (staphyloccin), staphyloccin (bacicin), staphylin (bacicin), bacicin (bacicin) (staphylin), bacicin (bacicin), bacicin (bacilin), bacilin (bacilin), bacil, Vibriocin (vibriocin), Wasserin (warnericin), cytolysin (cytolisin), pyocin S2(pyocyn S2), colicin A (colicin A), colicin E1(colicin E1), microcin McE 492(microcin McE 492), and warfarin (warnerin).

In some embodiments, the bacteriocin is obtained from gram-negative bacteria (e.g., microcins (e.g., microcin V from escherichia coli, subtilisin a (subtilosin a) from bacillus subtilis), colicins (e.g., colicins produced by escherichia coli and toxic to certain strains of escherichia coli (e.g., colicin a, colicin B, colicin E1, colicin E3, colicin E5, and colicin E7), tylosin (talilocin) (e.g., R-type pyocins, F-type pyocins)).

In some embodiments, the bacteriocin is obtained from a gram-positive bacterium (e.g., a class I bacteriocin (e.g., nisin, lantibiotic), a class II bacteriocin (e.g., ila-pediocin-like bacteriocin, lib bacteriocin (e.g., lactocin G), IIc cyclic peptide (e.g., enterococcin AS-48), IId single peptide bacteriocin (e.g., aureomycin a53), a class III bacteriocin (e.g., IIIa (e.g., lysin) and IIIb (which kills targets by disrupting membrane potential), or a class IV bacteriocin (e.g., complex bacteriocins containing lipid or carbohydrate moieties)).

Any of the bacterial systems described herein can further include a lytic plasmid and an activator plasmid. The lytic plasmid and the activator plasmid may each independently comprise any suitable components. The lytic and activator plasmids may be based on any suitable existing plasmid.

The lytic plasmid may comprise a lytic gene, an activatable promoter, and optionally a reporter gene. In some embodiments, the lytic gene is operably linked to an activatable promoter. In some embodiments, the reporter gene is operably linked to an activatable promoter. In some embodiments, both the lytic gene and the reporter gene are operably linked to a single copy of the activatable promoter. In some embodiments, both the lytic gene and the reporter gene are independently operably linked to separate copies of the activatable promoter.

The activator plasmid can include an activator gene, an optional degradation tag, and an optional reporter gene. In some embodiments, the activatable promoter in the activator plasmid is the same as the activatable promoter in the lytic plasmid. In some embodiments, the activatable promoter in the activator plasmid is activated by the same mechanism (e.g., directly or indirectly, through the same quorum sensing molecule) as the activatable promoter in the cleavage plasmid. In some embodiments, the activator gene can promote the accumulation of the quorum sensing molecule (e.g., the activator gene can encode a protein in the biosynthetic pathway of the quorum sensing molecule). In some embodiments, the activator gene is operably linked to an activatable promoter.

The quorum sensing molecule can be any suitable quorum sensing molecule. In some embodiments, the quorum sensing molecule may be N-Acyl Homoserine Lactone (AHL). In some embodiments, the quorum sensing molecule may be homoserine lactone (HSL) or a homolog thereof. In some embodiments, the quorum sensing molecule may be an autoinducing peptide (AIP) from a gram positive bacterium. Other quorum sensing molecules are known in the art.

The activatable promoter may be any suitable activatable promoter. In some embodiments, the activatable promoter may be activated directly or indirectly by a quorum sensing molecule. Thus in some embodiments, a feedback loop may be established such that the presence of quorum sensing molecules drives the accumulation of quorum sensing molecules. In some embodiments of any of the bacterial strains described herein, the activatable promoter is an AHL activatable promoter. In some embodiments of any of the bacterial strains described herein, the activatable promoter is a LuxR-AHL activatable promoter. In some embodiments, the activatable promoter is an RpaR-AHL activatable RpaI promoter. In some embodiments, the activatable promoter is a Trar-HSL activatable traI promoter. Other activatable promoters are known in the art.

The lytic gene may be any suitable lytic gene. In some embodiments of any of the bacterial strains described herein, the lytic gene is E from bacteriophage Φ X174. In some embodiments, the lytic gene is a colE1 lytic protein. Other lytic genes are known in the art.

The activator gene can be any suitable activator gene. In some embodiments, the activator gene is LuxI. In some embodiments, the activator gene is RpaI. In some embodiments, the activator gene is TraI. Other activator genes are known in the art.

In some embodiments, the quorum sensing molecule is AHL, the activatable promoter is a LuxR-AHL activatable luxI promoter, and the activator gene is luxI.

In some embodiments, the quorum sensing molecule is AHL, the activatable promoter is a RpaR-AHL activatable RpaI promoter, and the activator gene is RpaI.

In some embodiments, the quorum sensing molecule is HSL, the activatable promoter is a TraR-HSL activatable traI promoter, and the activator gene is traI.

The reporter gene may be any suitable reporter gene. In some embodiments, the reporter gene can be a fluorescent protein (e.g., Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), or a variant thereof). In some embodiments, the reporter gene can be an enzyme (e.g., β -galactosidase). Other reporter genes are known in the art.

The degradation label can be any suitable degradation label. In some embodiments, the degradation tag is an ssrA-LAA degradation tag. Other degradation tags are known in the art.

In some embodiments, the bacterial strain may encode additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins (e.g., payloads)) operably linked to a promoter-in some embodiments, the additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins) operably linked to a promoter are encoded on a plasmid. The plasmid may be any suitable plasmid. In some embodiments, the plasmid is a lytic plasmid or an activator plasmid. The additional nucleic acids and/or proteins may be any suitable nucleic acids and/or proteins. For example, the additional nucleic acid and/or protein may be a therapeutic agent. Any suitable promoter may be used. In some embodiments, the promoter may be an activatable promoter. In some embodiments, the promoter may be one that is activated directly or indirectly by the quorum sensing molecule. In some embodiments, the promoter can be an activatable promoter as described herein. In some embodiments, the promoter may be a constitutive promoter. Many constitutive promoters are known in the art. The therapeutic agent can be any suitable therapeutic agent. In some embodiments, the therapeutic agent can be any of the therapeutic agents described herein. For example, in some embodiments, the therapeutic agent may be selected from the group consisting of: inhibitory nucleic acids (e.g., siRNA, shRNA, miRNA or antisense (e.g., antisense DNA, antisense RNA or synthetic analogs)), cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, and antibodies or antigen-binding fragments thereof. In some embodiments, additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins) may be secreted, or otherwise exported from the bacterial strain, as described herein. In some embodiments, additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins) can be released upon lysis of the bacterial strain, as described herein. In some embodiments, additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins) can be displayed on the surface of the bacterial strain, as described herein.

In some embodiments, the additional nucleic acids and/or proteins (e.g., payloads) encoded by the bacterial strain may act on the additional nucleic acids and/or proteins (or products thereof) encoded by a previous (e.g., former) bacterial strain in a triple-or multi-lytic system. In some embodiments, the payload of the mth bacterial strain (e.g., one of the n bacterial strains) produces the mth substrate by acting directly or indirectly on the substrate of the (m-1) th bacterial strain, and wherein the payload of the first bacterial strain can act on the substrate present in the environment in which the n bacterial strains are cultured.

Provided herein are systems comprising two or more (e.g., three or more) bacterial strains described herein. In some embodiments, the system is a multi-lysis system. In some embodiments, the system may include 3 bacterial strains. In some embodiments, such systems may include 4, 5, 6, 7, 8, 9, 10, or more bacterial strains.

In some embodiments, when an activator plasmid/lytic plasmid system is used, the quorum sensing molecule for each bacterial strain in the system can be different. In some embodiments, the quorum sensing molecule of each of the bacterial strains in the system may have no or substantially no effect on the activatable promoter of the lytic gene of the other strain. In some embodiments, the quorum sensing molecule may be the same in each of the bacterial strains. In some embodiments, the lytic plasmid of each strain may be a copy of the same plasmid. In some embodiments, the activator plasmid of each bacterial strain may be a copy of the same plasmid. In some embodiments, the lytic plasmid of each bacterial strain may be a different plasmid. In some embodiments, the activator plasmid of each bacterial strain may be a different plasmid. In some embodiments, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) bacterial strains can be metabolically competitive. In some embodiments, the bacterial strain may be selected from escherichia coli, salmonella typhimurium, or a bacterial variant thereof. In some embodiments, none of the bacterial strains has a growth advantage over another strain in the system.

In some systems that include more than one bacterial strain, all of the bacterial strains may encode additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins). In some systems that include more than one bacterial strain, no bacterial strain may encode additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins). In some systems that include more than one bacterial strain, some bacterial strains may encode additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins), while others may not. In a system that includes multiple bacterial strains that encode additional nucleic acids and/or proteins (e.g., heterologous nucleic acids and/or proteins), the additional nucleic acids and/or proteins encoded by each such bacterial strain are independently selected (e.g., all such bacterial strains may encode different additional nucleic acids and/or proteins, all such bacterial strains may encode the same additional nucleic acids and/or proteins, or some such bacterial strains may encode the same additional nucleic acids and/or proteins, others encode one or more different additional nucleic acids and/or proteins).

Multiple lysis systems may include an appropriate number of strains. A triple lysis system may comprise three strains (this may also be referred to as "stone scissors cloth" (RPS) ecology). In such a system, each strain can encode a pair of toxin/antitoxin and a second antitoxin, such that the strain can kill a previous strain in a cycle (e.g., the second antitoxin encoded by the previous strain is ineffective against the toxin of the strain, and the second antitoxin encoded by the strain is effective against the toxin of the previous strain) and be killed by a subsequent strain in a cycle (e.g., the second antitoxin encoded by the strain is ineffective against the toxin of the subsequent strain, and the second antitoxin encoded by the subsequent strain is effective against the second toxin of the strain. an exemplary three-cleavage system is shown in fig. 12B.

In some embodiments, the multi-lytic system can include at least three strains (e.g., at least four, five, six, or more strains). In some embodiments, the multi-lytic system can have n strains, including a first strain, a second strain, and an nth strain. In some such cases, the second through nth strains may each have a previous strain based on the strain number (e.g., the previous strain of the second strain is the first strain). In some such cases, each of the second through n-1 strains has a subsequent strain (e.g., a subsequent strain of the n-1 strain is the nth strain). In some such cases, each of the second through nth strains encodes a first toxin effective against the previous strain, a first antitoxin against the first toxin, and a second antitoxin against a toxin produced by the previous strain. In some such cases, the first strain encodes a first toxin effective against the nth strain, a first antitoxin to the first toxin, and a second antitoxin to a toxin produced by the nth strain. The value of n may be any suitable value. In some embodiments, n can be at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

Also provided herein are methods of co-culturing at least three (e.g., at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) bacterial strains (e.g., any of the bacterial strains described herein). In some embodiments, co-culturing can include initially inoculating a culture with at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) bacterial strains (e.g., any of the bacterial strains described herein) at an inoculation ratio. The ratio of inoculation may be any suitable ratio. In some embodiments, the inoculation ratio of at least one bacterial strain to at least one other bacterial strain may be 1:1 (e.g., the ratio may be 1:1:1 for three strains). In some embodiments, the inoculation ratio of at least one bacterial strain to at least one other bacterial strain may be 1:2 (e.g., for three strains, the ratio may be 1:2: m,2:1: m,1: m:2,2: m:1, m:1:2, m:2: 1). In some embodiments, the inoculation ratio of at least one bacterial strain to at least one other bacterial strain may be 1:5 (e.g., for three strains, the ratio may be 1:5: m,5:1: m,1: m:5,5: m:1, m:1:5, m:5: 1). In some embodiments, the inoculation ratio of at least one bacterial strain to at least one other bacterial strain may be 1:10 (e.g., for three strains, the ratio may be 1:10: m,10:1: m,1: m:10,10: m:1, m:1:10, m:10: 1). The value of m may be any suitable value. For example, in some embodiments, m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

In some embodiments, co-culturing may comprise, in sequence, initiating culturing with a first bacterial strain, culturing for a period of time, adding a second bacterial strain, and culturing for a period of time. As used herein, the order may include a particular order of culturing two or more strains. In some embodiments, the sequence may include periods that are completely overlapping (e.g., growing periods), partially overlapping periods, and/or non-overlapping periods. In some embodiments, one or more growth phases may overlap, while other growth phases in the same system may not overlap. In some embodiments, all growth phases in a given system may or may not overlap. In some embodiments, the one or more additional bacterial strains may be cultured sequentially in the same manner as the second bacterial strain. In this way, sequential co-cultivation can be carried out in partially overlapping growth phases. In some embodiments, a first bacterial strain may be added to a culture containing a second bacterial strain and cultured for a period of time, and optionally taken over by the second bacterial strain and the first bacterial strain in an alternating manner. Thus, alternate co-cultures can be performed in partially overlapping growth phases. Addition of the bacterial strain may be performed at any suitable inoculation ratio based on the population of cells in the culture. For example, in some embodiments, the seeding ratio may be any of the seeding ratios described herein.

In some embodiments, co-culturing may comprise, in order, beginning the culturing with the first bacterial strain, culturing for a period of time, adding the second bacterial strain, and culturing for a period of time, adding the third bacterial strain, and culturing for a period of time. In some embodiments, the one or more additional bacterial strains may be cultured sequentially in the same manner as the second bacterial strain. In this way, sequential co-cultivation can be carried out in partially overlapping growth phases. In some embodiments, the cyclic co-cultivation with partially overlapping growth phases may be performed by adding the first bacterial strain to a culture containing the third bacterial strain (or one or more additional bacterial strains), culturing for a period of time, and then sequentially adding and culturing the second bacterial strain, the third bacterial strain, and the one or more additional bacterial strains in the same order as the original. Addition of the bacterial strain can be performed at any suitable inoculation ratio, where the inoculation ratio is based on the number of cells in the culture. For example, in some embodiments, the seeding ratio may be any of the seeding ratios described herein.

In some embodiments, the bacterial strain added to the culture may reduce or eliminate the population of one or more bacterial strains present in the culture. For example, in some embodiments, the second bacterial strain may reduce the population of the first bacterial culture or eliminate the first bacterial culture.

In some embodiments, the culture period (e.g., growth period) of any of the bacterial strains described herein can be from 1 hour to 35 days (e.g., from 1 hour to 30 days, from 1 hour to 28 days, from 1 hour to 26 days, from 1 hour to 25 days, from 1 hour to 24 days, from 1 hour to 22 days, from 1 hour to 20 days, from 1 hour to 18 days, from 1 hour to 16 days, from 1 hour to 14 days, from 1 hour to 12 days, from 1 hour to 10 days, from 1 hour to 8 days, from 1 hour to 7 days, from 1 hour to 6 days, from 1 hour to 5 days, from 1 hour to 4 days, from 1 hour to 72 hours, from 1 hour to 70 hours, from 1 hour to 68 hours, from 1 hour to 66 hours, from 1 hour to 64 hours, from 1 hour to 62 hours, from 1 hour to 60 hours, from 1 hour to 58 hours, from 1 hour to 56 hours, from 1 hour to 54 hours, from 1 hour to 52 hours, from 1 hour to 50 hours, from, 1 hour to 48 hours, 1 hour to 46 hours, 1 hour to 44 hours, 1 hour to 40 hours, 1 hour to 38 hours, 1 hour to 36 hours, 1 hour to 34 hours, 1 hour to 32 hours, 1 hour to 30 hours, 1 hour to 28 hours, 1 hour to 26 hours, 1 hour to 24 hours, 1 hour to 22 hours, 1 hour to 20 hours, 1 hour to 18 hours, 1 hour to 16 hours, 1 hour to 14 hours, 1 hour to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 4 hours, 1 hour to 2 hours, 2 hours to 35 days, 2 hours to 30 days, 2 hours to 28 days, 2 hours to 26 days, 2 hours to 25 days, 2 hours to 24 days, 2 hours to 22 days, 2 hours to 20 days, 2 hours to 18 days, 2 hours to 16 days, 2 hours to 14 days, 2 hours to 12 days, 2 hours to 10 days, 2 hours to 8 days, 2 hours to 7 days, 2 hours to 6 days, 2 hours to 5 days, 2 hours to 4 days, 2 hours to 72 hours, 2 hours to 70 hours, 2 hours to 68 hours, 2 hours to 66 hours, 2 hours to 64 hours, 2 hours to 62 hours, 2 hours to 60 hours, 2 hours to 58 hours, 2 hours to 56 hours, 2 hours to 54 hours, 2 hours to 52 hours, 2 hours to 50 hours, 2 hours to 48 hours, 2 hours to 46 hours, 2 hours to 44 hours, 2 hours to 40 hours, 2 hours to 38 hours, 2 hours to 36 hours, 2 hours to 34 hours, 2 hours to 32 hours, 2 hours to 30 hours, 2 hours to 28 hours, 2 hours to 26 hours, 2 hours to 24 hours, 2 hours to 22 hours, 2 hours to 20 hours, 2 hours to 18 hours, 2 hours to 16 hours, 2 hours to 14 hours, 2 hours to 12 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 2 hours to 4 hours, 4 hours to 35 days, 4 hours to 30 days, 4 hours to 28 days, 4 hours to 26 days, 4 hours to 25 days, 4 hours to 24 days, 4 hours to 22 days, 4 hours to 20 days, 4 hours to 18 days, 4 hours to 16 days, 4 hours to 14 days, 4 hours to 12 days, 4 hours to 10 days, 4 hours to 8 days, 4 hours to 7 days, 4 hours to 6 days, 4 hours to 5 days, 4 hours to 4 days, 4 hours to 74 hours, 4 hours to 70 hours, 4 hours to 68 hours, 4 hours to 66 hours, 4 hours to 64 hours, 4 hours to 60 hours, 4 hours to 58 hours, 4 hours to 56 hours, 4 hours to 54 hours, 4 hours to 54 hours, 4 hours to 50 hours, 4 hours to 48 hours, 4 hours to 46 hours, 4 hours to 44 hours, 4 hours to 40 hours, 4 hours to 38 hours, 4 hours to 36 hours, 4 hours to 34 hours, 4 hours to 30 hours, 4 hours to 28 hours, 4 hours to 26 hours, 4 hours to 24 hours, 4 hours to 20 hours, 4 hours to 18 hours, 4 hours to 16 hours, 4 hours to 14 hours, 4 hours to 10 hours, 4 hours to 8 hours, 4 hours to 6 hours, 6 hours to 35 days, 6 hours to 30 days, 6 hours to 28 days, 6 hours to 26 days, 6 hours to 25 days, 6 hours to 24 days, 6 hours to 22 days, 6 hours to 20 days, 6 hours to 18 days, 6 hours to 16 days, 6 hours to 14 days, 6 hours to 12 days, 6 hours to 10 days, 6 hours to 8 days, 6 hours to 7 days, 6 hours to 6 days, 6 hours to 5 days, 6 hours to 4 days, 6 hours to 76 hours, 6 hours to 70 hours, 6 hours to 68 hours, 6 hours to 66 hours, 6 hours to 64 hours, 6 hours to 66 hours, 6 hours to 60 hours, 6 hours to 58 hours, 6 hours to 56 hours, 6 hours to 54 hours, 6 hours to 56 hours, 6 hours to 50 hours, 6 hours to 48 hours, 6 hours to 46 hours, 6 hours to 44 hours, 6 hours to 40 hours, 6 hours to 38 hours, 6 hours to 36 hours, 6 hours to 34 hours, 6 hours to 36 hours, 6 hours to 30 hours, 6 hours to 28 hours, 6 hours to 26 hours, 6 hours to 24 hours, 6 hours to 26 hours, 6 hours to 20 hours, 6 hours to 18 hours, 6 hours to 16 hours, 6 hours to 14 hours, 6 hours to 16 hours, 6 hours to 10 hours, 6 hours to 8 hours, 12 hours to 35 days, 12 hours to 30 days, 12 hours to 28 days, 12 hours to 26 days, 12 hours to 25 days, 12 hours to 24 days, 12 hours to 22 days, 12 hours to 20 days, 12 hours to 18 days, 12 hours to 16 days, 12 hours to 14 days, 12 hours to 12 days, 12 hours to 10 days, 12 hours to 8 days, 12 hours to 7 days, 12 hours to 6 days, 12 hours to 5 days, 12 hours to 4 days, 12 hours to 72 hours, 12 hours to 70 hours, 12 hours to 68 hours, 12 hours to 66 hours 12 hours to 64 hours, 12 hours to 62 hours, 12 hours to 60 hours, 12 hours to 58 hours, 12 hours to 56 hours, 12 hours to 54 hours, 12 hours to 512 hours, 12 hours to 50 hours, 12 to 48 hours, 12 to 46 hours, 12 to 44 hours, 12 to 40 hours, 12 to 38 hours, 12 to 36 hours, 12 to 34 hours, 12 to 312 hours, 12 to 30 hours, 12 to 28 hours, 12 to 26 hours, 12 to 24 hours, 12 to 22 hours, 12 to 20 hours, 12 to 18 hours, 12 to 16 hours, 12 to 14 hours, 1 to 35 days, 1 to 30 days, 1 to 28 days, 1 to 26 days, 1 to 25 days, 1 to 24 days, 1 to 22 days, 1 to 20 days, 1 to 18 days, 1 to 16 days, 1 to 14 days, 1 to 12 days, 1 to 10 days, 1 to 8 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 35 days, 2 to 30 days, 2 days, 1 to 8 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 30 days, 2 to 30 days, 1 to 10 days, 1 to 25 days, 1 day, 1 to 25, 2 to 28 days, 2 to 26 days, 2 to 25 days, 2 to 24 days, 2 to 22 days, 2 to 20 days, 2 to 18 days, 2 to 16 days, 2 to 15 days, 2 to 14 days, 2 to 12 days, 2 to 10 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, 2 to 3 days, 4 to 35 days, 4 to 30 days, 4 to 28 days, 4 to 26 days, 4 to 25 days, 4 to 24 days, 4 to 22 days, 4 to 20 days, 4 to 18 days, 4 to 16 days, 4 to 15 days, 4 to 14 days, 4 to 12 days, 4 to 10 days, 4 to 8 days, 4 to 6 days, 7 to 35 days, 7 to 30 days, 7 to 28 days, 7 to 26 days, 7 to 25 days, 7 to 7 days, 7 to 15 days, 7 days, 2 to 10 days, 2 to 8 days, 2 days, 4 to 8 days, 4 days, 3 days, 4 to 35 days, 7 days, 7 to 14 days, 7 to 12 days, 7 to 10 days, 7 to 8 days, 14 to 35 days, 14 to 30 days, 14 to 28 days, 14 to 26 days, 14 to 25 days, 14 to 24 days, 14 to 22 days, 14 to 20 days, 14 to 18 days, 14 to 16 days, 14 to 15 days, 21 to 35 days, 21 to 30 days, 21 to 28 days, 21 to 26 days, 21 to 25 days, 21 to 24 days, 21 to 22 days, 28 to 35 days, or 28 to 30 days; 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 22 days, 24 days, 25 days, 26 days, 28 days, 30 days, 32 days, 34 days, or 35 days).

The length of the cycle can be adjusted by any suitable means. For example, the length of the cycle can be adjusted by using strains that lyse at different ODs. Cell lysis can also be regulated by modulating the internal circuitry of the quorum sensing component, e.g., modulating AHL degradation, modulating cleavage of protein degradation, modulating a promoter to increase or decrease expression of molecules involved in the quorum sensing circuit.

Various methods known in the art may be employed to determine whether a population threshold has been reached. For example, the population threshold can be determined by measuring the expression level of AHL in the culture medium using conventional protein quantification methods. Population thresholds can also be determined using reporter proteins driven by the luxI promoter. In some embodiments, the reporter protein is a fluorescent protein, a bioluminescent luciferase reporter, a secreted blood/serum or urine reporter (e.g., secreted alkaline phosphatase, soluble peptide, gauss luciferase).

Various methods are known in the art to determine and/or measure cell lysis. For example, cell lysis can be phenotypically determined microscopically by changes in intensity of transmitted and/or absorbed light of various wavelengths, including light at 600 nm. In some embodiments, bacterial cell lysis is synchronized. In other embodiments, bacterial cell lysis is asynchronous. Simultaneous lysis can be achieved by passing the absorption Optical Density (OD) at 600nm in a plate reader or other quantification device ₆₀₀) To be measured.

In some embodiments of any of the bacterial strains described herein, the therapeutic agent is selected from the group consisting of: inhibitory nucleic acids (e.g., siRNA, shRNA, miRNA, or antisense nucleic acids), cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins (e.g., diphtheria toxin, gelonin, anthrax toxin), antimicrobial peptides, and antibodies or antigen-binding fragments thereof. Non-limiting examples of cytokines include filgrastim, interleukins, interferons (e.g., IFNb-1b), and transforming growth factor beta. Non-limiting examples of enzymes include asparaginase, glucosidase, β -glucocerebrosidase, elosulfatase (elsufase) α, asportase (aspotase) α, and seebeclipase (sebispase) α. Non-limiting examples of peptide hormones include metreleptin, parathyroid hormone and insulin. Non-limiting examples of fusion proteins include Albiglutide, dolaglutide, factor IX Fc fusion, and factor VIII Fc fusion. Non-limiting examples of blood coagulation factors include factor IX, factor VIII, and factor XIII a subunits. Non-limiting examples of the antibody or antigen-binding fragment thereof include belimumab (belimumab), ipilimumab (ipilimumab), belicept (belicept), benazemab (bentazaept), bentuximab (brentuximab), aflibercept (afilbercept), pertuzumab (pertuzumab), resisibutrumab (raxibacumab), trastuzumab (trastuzumab), homolizumab (golimab), bevacizumab (bevacizumab), oriuzumab (obiuzumab), ramucirumab (ramucimab), trastuzumab (siltuzumab), rituximab (rituximab), adalimumab (adalimumab), vedolizumab (vedolizumab), pembrolizumab (pemirolimumab), pembrolizumab (blevacizumab), polimumab (edalimumab), vezumab (vezumab), pemumab (blevacizumab), pemutalizumab (blevacizumab), pemutab (blevacizumab), polivacizumab (polivacizumab), pemutab (blevacizumab), pemetrexelbutab (blevacizumab), pembrolizumab (e (niuzumab), pemetrexelizumab), pembrolizumab), pemetrexendin (e), pembrolizumab), pemetrexex (e), bevacizumab), pembrolizumab), pembro, Erlotinib (elotuzumab), obiteximab (obilitoxaximab), ixlizumab (ixekizumab), rayleigh-lizumab (resilizumab), infliximab (infliximab), azilizumab (atezolizumab), dallizumab (daclizumab), ubuzumab (usekinumab), and etanercept (etanercept). In some embodiments, the therapeutic agent may be a C1 esterase inhibitor. In some embodiments, the therapeutic agent can be von willebrand factor.

Provided herein are pharmaceutical compositions comprising any one or more bacterial strains as described herein. In some embodiments, the pharmaceutical composition is formulated for in situ drug delivery.

Also provided herein are methods that can include co-culturing at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the bacterial strains described herein.

Also provided herein are pharmaceutical systems comprising one or more of the systems described herein.

Also provided herein are periodic drug systems comprising one or more of the systems described herein.

Also provided herein are kits comprising one or more of the bacterial strains described herein.

Also provided herein are kits comprising one or more of the systems described herein.

Provided herein are methods of treating a disease (e.g., cancer, infectious disease, autoimmune disease, genetic disease, metabolic disease) in a subject. An exemplary method comprises administering to a subject in need of treatment a therapeutically effective amount of any one or more of the bacterial strains described herein, or any one or more of the pharmaceutical compositions described herein, or any one or more of the systems described herein, thereby treating a disease in the subject. For example, provided herein is a method of treating a disease in a subject comprising administering to the subject a therapeutically effective amount of each of n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain. Each of the second to nth strains may have a previous bacterial strain. The value of n may be at least 3. Each of the n strains may comprise a toxin system as described herein. In certain instances, the toxin system of each of the second through nth strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous strain, wherein the first toxin is effective against the previous strain. The toxin system of the first strain can produce a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth strain, wherein the third toxin is effective against the nth strain. In some embodiments, one or more of the n strains can include a lytic plasmid described herein and/or an activator plasmid described herein. The value of n may be any suitable value. In some embodiments, n can be at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

Provided herein are methods of treating a disease (e.g., cancer, infectious disease, autoimmune disease, genetic disease, metabolic disease) in a subject. An exemplary method comprises administering to a subject in need of treatment a therapeutically effective amount of a pharmaceutical composition described herein comprising any one or more of the bacterial strains described herein, or any one or more of the pharmaceutical compositions described herein, or any one or more of the systems described herein, thereby treating a disease in the subject. For example, provided herein is a method of treating a disease in a subject comprising administering to the subject a therapeutically effective amount of m pharmaceutical compositions, each comprising one or more of n bacterial strains, including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain. Each of the second to nth strains may have a previous bacterial strain. The value of n may be at least 3. The value of m may be at least 3. The value of m may be equal to the value of n. Each of the n strains may include a toxin system as described herein. In certain instances, the toxin system of each of the second through nth strains independently produces a first toxin/first antitoxin pair, and a second antitoxin from a second toxin/second antitoxin pair produced by a previous strain, wherein the first toxin is effective against the previous strain. The toxin system of the first strain can produce a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth strain, wherein the third toxin is effective against the nth strain. In some embodiments, one or more strains of n can include a lytic plasmid described herein and/or an activator plasmid described herein. The value of n may be any suitable value. In some embodiments, n can be at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). The value of m may be any suitable value. In some embodiments, m can be at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

Administration may include any suitable schedule. For example, administration may include a schedule effective for co-culturing as described herein. In some embodiments, as described herein, administration may include a schedule effective for co-culturing including partially overlapping growth phases. In the methods described herein, administering can include administering at least two (e.g., at least three) bacterial strains to the subject.

Also provided herein are methods of treating a disease (e.g., cancer, infectious disease, autoimmune disease, genetic disease, metabolic disease) in a subject comprising administering to a subject in need of treatment a therapeutically effective amount of a first bacterial strain (e.g., any bacterial strain described herein) for a first period of time, a therapeutically effective amount of a second bacterial strain (e.g., any bacterial strain described herein) for a second period of time, and a therapeutically effective amount of a third bacterial strain (e.g., any bacterial strain described herein) for a third period of time.

In some embodiments of any of the methods described herein, the n bacterial strains (e.g., the first, second, and third bacterial strains) are different bacterial strains, each expressing and/or secreting a different therapeutic agent (e.g., any of the therapeutic agents described herein). In some embodiments of any of the methods described herein, the n bacterial strains (e.g., the first, second, and third bacterial strains) are different bacterial strains, each expressing and/or secreting the same therapeutic agent (e.g., any of the therapeutic agents described herein). In some embodiments of any of the methods described herein, the n bacterial strains (e.g., the first, second, and third bacterial strains) are different bacterial strains that may or may not each express and/or secrete the same or different therapeutic agent (e.g., any of the therapeutic agents described herein) (e.g., two strains may express and/or secrete one therapeutic agent and a third strain may express and/or secrete a different therapeutic agent). The value of n may be any suitable value. In some embodiments, n can be at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

In some embodiments of any of the methods described herein, the n bacterial strains (e.g., the first, second, and third bacterial strains) do not express or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein). In some embodiments of any of the methods described herein, the n bacterial strains (e.g., the first, second, and third bacterial strains) produce a bacteriocin (e.g., any of the bacteriocins described herein). The value of n may be any suitable value. In some embodiments, n can be at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

In some embodiments of any of the methods described herein, the administering comprises a treatment cycle, and the treatment cycle is repeated at least twice (e.g., at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times).

In some embodiments of any of the methods described herein, the first, second, and third time periods can be between 1 hour and 35 days (e.g., between 1 hour and 28 days, between 1 hour and 21 days, between 1 hour and 14 days, between 1 hour and 7 days, between 1 hour and 5 days, between 1 hour and 3 days, between 1 hour and 2 days, between 1 hour and 1 day, between 1 day and 35 days, between 1 day and 28 days, between 1 day and 21 days, between 1 day and 14 days, between 1 day and 7 days, between 1 day and 5 days, between 1 day and 3 days, between 1 day and 2 days, between 7 days and 35 days, between 7 days and 28 days, between 7 days and 14 days, between 7 days and 10 days, between 14 days and 35 days, between 14 days and 28 days, between 14 days and 21 days, between 21 days and 35 days, between 21 days and 28 days, 21 days to 24 days or 28 days to 35 days).

In some aspects of any of the methods described herein, the first, second, and third time periods are similar in length, e.g., the same length of time.

In some aspects of any of the methods described herein, the first, second, and third time periods are different in length. In certain aspects, the first and third time periods are the same. In certain aspects, the first and third time periods are different. In certain aspects, the first and second time periods are the same. In certain aspects, the first and second time periods are different. In certain aspects, the second and third time periods are the same. In certain aspects, the second and third time periods are different.

In some embodiments of any of the methods described herein, the growth period can be between 1 hour and 35 days (e.g., between 1 hour and 28 days, between 1 hour and 21 days, between 1 hour and 14 days, between 1 hour and 7 days, between 1 hour and 5 days, between 1 hour and 3 days, between 1 hour and 2 days, between 1 hour and 1 day, between 1 day and 35 days, between 1 day and 28 days, between 1 day and 21 days, between 1 day and 14 days, between 1 day and 7 days, between 1 day and 5 days, between 1 day and 3 days, between 1 day and 2 days, between 7 days and 35 days, between 7 days and 28 days, between 7 days and 14 days, between 7 days and 10 days, between 14 days and 35 days, between 14 days and 28 days, between 14 days and 21 days, between 21 days and 35 days, between 21 days and 28 days, between 21 days and 24 days, or 28 days).

In some aspects of any of the methods described herein, the growth phase of each of the n bacterial strains is about the same. In some embodiments of any of the methods described herein, one or more of the n bacterial strains have a different growth phase than one or more of the other bacterial strains.

In some embodiments of any of the methods described herein, administering comprises administering the at least three bacterial strains to the subject separately and sequentially. In some embodiments of any of the methods described herein, administering comprises administering each of the n bacterial strains to the subject sequentially.

In some embodiments of any of the methods described herein, administering comprises administering each of the at least three bacterial strains simultaneously. In some embodiments of any of the methods described herein, administering comprises administering each of the n bacterial strains simultaneously.

In some embodiments of any of the methods described herein, the subject has cancer or an infection.

In some embodiments where the subject has cancer, the cancer may be, for example, a primary tumor or a metastatic tumor.

In some embodiments, the cancer is a non-T cell infiltrating tumor.

In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular carcinoma, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer. The present invention contemplates the simultaneous treatment of multiple cancer species and is encompassed by the present invention.

In some cases, the subject having cancer may have previously received a cancer treatment (e.g., a treatment for any of the cancers described herein).

In some embodiments of any of the methods described herein, the subject has an infection (e.g., an infectious disease). In some embodiments of any of the methods described herein, the infection is caused by an infectious agent selected from the group consisting of: campylobacter jejuni (Campylobacter jejuni), Clostridium botulinum (Clostridium botulinium), Escherichia coli (Escherichia coli), Listeria monocytogenes (Listeria monocytogenes), and Salmonella (Salmonella).

In some embodiments, strain a or a first strain is first introduced to a site of culture or colonization for a specific period of time to deliver its payload of a therapeutic protein. A second strain, strain B, can then be introduced to produce toxin killing and removal of strain a, and then strain B can deliver a payload of therapeutic protein over a specified period of time. A third strain, strain C, can then be introduced, killing and removing strain B, and then strain C can deliver a payload of therapeutic protein over a specified period of time. Strain a can then be reintroduced to repeat the cycle. The system is not limited to a three strain system and may consist of more than three strains, each of which produces a toxin capable of killing another strain in the system that is not antitoxin free, thereby forming a circulatory system (consisting of four, five, six or more strains).

Administration can be performed, e.g., at least once per week (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times). Treatment is also contemplated on a monthly basis, such as at least once a month, for at least 1 month (e.g., at least two, three, four, five or six months, or more months, such as 12 months or more), and on an annual basis (e.g., once a year, for one or more years). Administration can be by any means known in the art, such as intravenous, subcutaneous, intraperitoneal, oral, topical, and/or rectal administration, or any combination of known methods of administration.

As used herein, treatment includes "prophylactic treatment," which refers to reducing the incidence of a disease (e.g., cancer, infection) or preventing (or reducing the risk of) signs or symptoms of a disease (e.g., cancer, infection) in a subject at risk of developing the disease (e.g., cancer, infection). The term "therapeutic treatment" refers to reducing signs or symptoms of a disease, e.g., reducing cancer progression, reducing the severity of cancer and/or recurrence in a subject with cancer, reducing inflammation in a subject, reducing the spread of infection in a subject.

The recurrence described herein may be used for cancer treatment. Non-limiting examples of cancer include: acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), adrenocortical carcinoma, anal carcinoma, appendiceal carcinoma, astrocytoma, basal cell carcinoma, brain carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, breast carcinoma, bronchial carcinoma, Burkitt's lymphoma, carcinoma of unknown primary origin, cardiac tumor, cervical carcinoma, chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative tumor, colon carcinoma, colorectal carcinoma, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial carcinoma, ependymoma, esophageal carcinoma, sensory neuroblastoma, fibroblastic tumor, Ewing's sarcoma, eye carcinoma, germ cell tumor, gallbladder carcinoma, gastric carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational cell disease, glioma, head and neck carcinoma, neuroblastoma, colon carcinoma, Hairy cell leukemia, hepatocellular carcinoma, histiocytosis, hodgkin's lymphoma, hypopharynx cancer, intraocular melanoma, islet cell tumor, kaposi's sarcoma, kidney cancer, langerhans 'cell histiocytosis, larynx cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, merkel cell cancer, mesothelioma, metastatic squamous neck cancer with occult primary, midline cancer involving the NUT gene, mouth cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative tumors, nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, human melanoma, human immunodeficiency virus, human immunodeficiency, Papillomatosis, paraganglioma, parathyroid carcinoma, penile carcinoma, pharyngeal carcinoma, pheochromocytoma, pituitary cancer, pleuropneumocytoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdoid tumor, salivary gland carcinoma, sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, malformation tumor, testicular cancer, throat cancer, thymoma and thymus cancer, thyroid cancer, urinary tract cancer, uterine cancer, vaginal cancer, vulval cancer and wilm's tumor.

For example, any of the methods described herein can be used to treat a cancer selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular carcinoma, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer.

The term "therapeutic agent" refers to a therapeutic treatment involving the administration of a therapeutic agent to a subject that is known to be useful in the treatment of a disease (e.g., cancer, infection). For example, cancer therapeutics can reduce tumor size or tumor growth rate. In another instance, the cancer therapeutic can affect the tumor microenvironment.

Non-limiting examples of therapeutic agents that may be expressed and/or secreted in any of the bacterial strains described herein include: inhibitory nucleic acids (e.g., microrna (mirna), short hairpin rna (shrna), small interfering rna (sirna), antisense nucleic acids), cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins (e.g., diphtheria toxin, gelonin, anthrax toxin), antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

In some cases, the therapeutic agent is a therapeutic polypeptide. In some cases, the therapeutic polypeptide includes one or more (e.g., 2, 3, 4, 5, or 6) polypeptides. In some cases, the therapeutic polypeptide is conjugated to a toxin, radioisotope, or drug via a linker (e.g., cleavable linker, non-cleavable linker).

In some cases, the therapeutic agent is cytotoxic or cytostatic to the target cell.

The phrase "cytotoxic to target cells" refers to an induction in the death (e.g., necrosis or apoptosis) of the target cells, either directly or indirectly. For example, the target cell may be a cancer cell (e.g., a cancer cell or a tumor-associated immune cell (e.g., a macrophage) or an infected cell.

The phrase "cytostatic to a target cell" refers to a direct or indirect reduction in the proliferation (cell division) of the target cell in vivo or in vitro. Where the therapeutic agent is cytostatic to the target cell, the therapeutic agent may, for example, directly or indirectly cause cell cycle arrest of the target cell. In some embodiments, the cytostatic therapeutic agent can reduce the number of target cells in the S phase in the cell population (compared to the number of target cells in the S phase in the cell population between contact with the therapeutic agent). In some cases, the cytostatic therapeutic agent can reduce the percentage of target cells in S phase by at least (e.g., at least 40%, at least 60%, at least 80%) compared to the percentage of target cells in S phase in the cell population between contact with the therapeutic agent.

Also provided herein are pharmaceutical compositions comprising at least three (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) of any of the bacterial strains described herein that express and/or secrete at least one of any of the therapeutic agents described herein.

The pharmaceutical composition may be formulated in any manner known in the art. The pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, subcutaneous, intraperitoneal, rectal, topical, or oral). In some embodiments, the pharmaceutical composition is administered directly to the site of the disease or diseased tissue, e.g., to a tumor, to infected tissue. In some embodiments, administration is targeted, e.g., the pharmaceutical composition comprises a targeting moiety (e.g., a targeting protein or peptide).

In some embodiments, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier (e.g., phosphate buffered saline). The pharmaceutical composition may include any suitable additional agent, such as excipients, fillers, disintegrants, lubricants, glidants, binders, solubilizers, fillers, buffers, surfactants, chelating agents, adjuvants, and the like, as is known in the art. The formulation may be administered in a single or multiple doses depending on, for example: the dose (i.e., number of bacterial cells per ml) and the frequency required and tolerated by the subject. The dose, frequency and timing required to effectively treat a subject can be influenced by the age of the subject, the general health of the subject, the severity of the disease, previous treatments, and the presence of co-morbidities (e.g., diabetes, cardiovascular disease). The formulation should provide a sufficient amount of the active agent to effectively treat, prevent or alleviate the condition, disease or symptom. Toxicity and therapeutic efficacy of the compositions can be determined in cell cultures, preclinical models (e.g., mouse, rat, or monkey), and humans using conventional methods. The data obtained from in vitro testing and preclinical studies can be used to formulate an appropriate dosage of any of the compositions described herein (e.g., a pharmaceutical composition described herein).

The efficacy of any of the compositions described herein can be determined using methods known in the art, for example, by observing clinical signs of disease (e.g., tumor size, presence of metastases).

Also provided herein are kits comprising at least three of any of the bacterial strains described herein that express and/or secrete at least one of any of the therapeutic agents described herein. In some cases, the kit can comprise instructions for performing any of the methods described herein. In some embodiments, the kit can comprise at least one dose of any of the pharmaceutical compositions described herein. The kits described herein are not so limited; other variations will be apparent to persons skilled in the art.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention as claimed.

Materials and methods

Strains and plasmids.

Strains containing plasmids of ColE1 starting plasmid and p15A starting plasmid, respectively, as a loop strain in a medium containing 50. mu.g ml^-1Kanamycin and 34. mu.g ml^-1Culturing in broth (LB) containing chloramphenicol, adding 0.2% glucose, or for non-lytic colicin strain, 50 μ g ml ^-1The spectinomycin medium was cultured in a 37 ℃ shaking incubator. Exemplary toxins are shown in fig. 4 and 22. Exemplary strains are shown in fig. 5 and 23. Exemplary plasmids used in this study are described in fig. 11 and 21. The colicin E3 and E5 genes were assembled using gene block overlap PCR (IDT). The colicin V gene was obtained from the PCR off product of wild colicin V E.coli (obtained from doctor Joe Pogliano and Robert Kolter). All plasmids were constructed by Gibson assembly and then transformed into DH5 α (Thermo fisher) chemically competent e. The plasmid was verified by Sanger sequencing before transformation into e.coli MG1655 strain.

For toxin co-culture experiments, wild-type MG1655 E.coli strain was inoculated from-80 ℃ glycerol stock into 2ml LB, and cultured in a 37 ℃ shaking incubator. The OD between 0.2-0.4 is reached in the cells₆₀₀Thereafter, 5. mu.l of the culture was added to 200. mu.l of fresh medium in a standard Falcon tissue culture 96-well flat bottom plate. In addition, 5. mu.l of purified colicin lysate was added to each well. Cultures were grown at 37 ℃ for 19 hours with shaking and the optical density as indicated by absorbance at 600nm was measured every 5 minutes using Tecan Infinite M200 Pro. Fluorescence was measured once in 5 minutes, and GFP, CFP and RFP were measured at 485nm excitation emission at 520nm, 433nm excitation at 475nm and 590nm excitation at 630nm, respectively.

Microfluidics and microscope

The microfluidic devices and experimental preparation protocols used in this study were similar to those reported elsewhere (see, e.g., Prandle et al, A sensing array of radial coupled genetic biopixels (a sensor array consisting of genetic biological samples) Nature, 481 (7379): 39-442012). What is needed isSome microfluidic experiments were performed in a side-well array device with a bacterial growth chamber area of about 100x80 μm and a height of about 1.2 μm. The appropriate E.coli strain was inoculated from a-80 ℃ glycerol stock into 5ml LB and the appropriate antibiotic was added. To lyse the strain, 0.2% glucose was added to the medium. After 8-12 hours of growth at 37 ° in a shaking incubator, the cultures were diluted 100-fold into 25mL of the same medium, placed in 50mL Erlenmeyer flasks, and grown to an OD of between 0.4 and 0.6 (using a Plastibrand 1.5mL tube). Once the above OD is reached, the cells are concentrated by centrifugation at 5000g for 1 minute and resuspended in 10. mu.l of LB medium containing 0.75% Tween-20 and the appropriate antibiotic. The concentrate was used for cell vacuum loading for single strain assays. For the multiple strain experiments, cultures were first normalized to the same OD prior to centrifugation ₆₀₀And mixed at a ratio of 1:1, 1:2, 4:6 or 1:5 in 2 strain experiments or at a ratio of 1:1:1 before vacuum loading for 3 strain experiments. The control of the flow of media into the microfluidic device is gravity driven. The chip temperature was maintained at 37 ℃ and the entire microscope was held in a plexiglas incubator.

For successive loading experiments, two initial strains were prepared as described for the 2 strain experiment. The subsequent strain was inoculated from a-80 ℃ glycerol stock into 5ml LB supplemented with 0.2% glucose and appropriate antibiotics. After 8-12 hours of growth at 37 ℃ in a shaking incubator, the cultures were diluted 100-fold into 100mL of the same medium, placed in 500mL Erlenmeyer flasks, and grown to OD between 0.1-0.4 (using a Plastibrand 1.5mL tube). Once the above OD is reached, the cells are concentrated by centrifugation at 5000g for 5 minutes and resuspended in 1ml of LB medium containing 0.075% Tween-20 and the appropriate antibiotic. The cells are then added to the second or third inlet of the microfluidic device and introduced into the well by flick loading. Flick loading is accomplished by flicking the Tygon tube to the inlet of the microfluidic device in order to disrupt laminar flow within the device, allowing the cells to be washed into the wells of the microfluidic. The media flow control of microfluidic devices is gravity driven. During the experiment, a solution containing 34. mu.g ml of the reagent was used ^-1LB medium for chloramphenicol. Keeping the temperature of the chip at 37 deg.C, using organic solventThe glass incubator houses the entire microscope.

Nikon (Nikon) Eclipse TI epifluorescence microscopy with phase contrast imaging was used. Image acquisition was done with Photometrics CoolSnap cooled CCD camera and Nikon Elements software. For 10-fold magnification experiments, phase contrast images were taken at 50-100 μ s exposure time. The fluorescence exposure times for gfp, rfp and cfp were 100 μ s at 30% intensity, 300 μ s at 30% intensity and 100 μ s at 30% intensity, respectively. Images were taken every 3 minutes or every 6 minutes during the experiment (1-4 days). For the 60-fold magnification experiment, the phase difference image was taken at an exposure time of 20-50 μ s. The fluorescence exposure times for gfp, rfp and cfp were 50 μ s at 30% intensity, 200 μ s at 30% intensity and 50 μ s at 30% intensity, respectively. Images were taken every 3 minutes or every 6 minutes during the experiment (1-4 days). For sequential loading experiments, the shooting was suspended during the loading of the strains.

Population estimation and growth rate of RPS strains.

Population estimation of the large population fraction in the co-cultivation mixture was performed in the following manner. The fluorescence channel image stack from the dominant strain was converted to 8 bits in ImageJ (treatment > binary > generation binary). A Z-axis cross-sectional view of the cell incubation area (image > stacked Z-axis profile) is then taken and the value normalized to 1 by dividing by the maximum value. The maximum value of the fluorescence time series trace of the dominant strain was taken as the cumulative fluorescence standard for the individual cultures of the 100% dominant strain. The susceptible strain share was estimated by subtracting the normalized dominant strain value from 1. These values were then normalized to the initial cell fraction corresponding to approximately 70% of the dominant strain and 30% of the susceptible strain. Initial cell shares were calculated manually using the ImageJ cell count insert.

For growth rate experiments, appropriate E.coli strains were inoculated from-80 ℃ glycerol stocks into 2ml LB and appropriate antibiotics and cultured in a 37 ℃ shake incubator. OD of 0.1 was achieved in the cells₆₀₀Thereafter, 1ml of culture was added to a 125ml Erlenmeyer flask containing 25ml of fresh medium and the appropriate antibiotic and shaken at 270 rpm. Once the sample reached an OD of 0.1₆₀₀Every 10 thSamples were collected once a minute and at OD using a DU 740Life Science UV/Vis spectrophotometer₆₀₀To perform the measurement.

Passage and plate reader experiment

For passage experiments, the appropriate E.coli strains were inoculated from a-80 ℃ glycerol stock into 2ml LB containing 0.2% glucose and the appropriate antibiotics and cultured in a shaking incubator at 37 ℃. At 0.2OD₆₀₀Next, 10. mu.l of the culture was added to 190. mu.l of fresh medium in a standard Falcon tissue culture 96-well flat bottom plate. Cells were cultured with shaking at 37 ℃ in Tecan Infine M200 Pro for 12 hours per subculture, and the optical density at absorbance of 600nm was measured every 10 minutes. Fluorescence was measured once in 10 minutes, and GFP, CFP and RFP were measured at 485nm excitation emission at 520nm, 433nm excitation at 475nm and 590nm excitation at 630nm, respectively. After 12 hours, each sample was diluted to OD ₆₀₀0.2 and 10. mu.l of each sample was passaged to 190. mu.l of fresh medium containing the appropriate antibiotic in a new plate. For circulating strains, appropriate E.coli strains were grown from-80 ℃ glycerol stocks to OD as described previously₆₀₀0.2, then passage immediately, followed by addition of 10 μ l of the culture containing the new strain to the appropriate passage before measurement. Sanger sequencing samples were collected at the end of each passage. For analysis, a successful cleavage event was defined as OD₆₀₀A curve was grown with a defined peak followed by a drop in OD.

Plate reader fluorescence

For well-plate experiments, appropriate E.coli strains were inoculated from-80 ℃ glycerol stocks into 2ml LB containing 0.2% glucose and appropriate antibiotics and cultured in a shaking incubator at 37 ℃. The OD between 0.2-0.6 is reached in the cells₆₀₀Thereafter, 5. mu.l of the culture was added to 200. mu.l of fresh medium containing the appropriate antibiotic in a standard Falcon tissue culture 96-well flat bottom plate. Cells were cultured at 37 ℃ with shaking and imaged every 5 minutes in Tecan Infit M200 Pro. The dilutions were grown for 19 hours and the optical density of absorbance at 600nm was measured every 5 minutes. The fluorescence was measured once in 5 minutes, respectively excited at 485nm at 520n GFP, CFP and RFP were measured at m-emission, 433nm excitation 475nm emission and 590nm excitation 630nm emission. The resulting curves were used to estimate the OD600 of lysis and growth kinetics for each strain.

Example 1 two Strain System

The first stage of development involves engineering a selective advantage. Strategies were initially investigated to confer a selective advantage on one strain over the others.

Strategies were initially investigated to confer a selective advantage on one strain over the others. From the range of possible colicins, two were identified (colicin E3, colicin E7) that were lethal by dnase and rRNA enzyme activities (see, e.g., fig. 4 and 22). The toxin effect was verified in microfluidic and plate reader experiments (fig. 5, fig. 6). To describe this effect, we constructed a p15A starting plasmid containing colicin and Col E1 cleavage protein driven by a weak constitutive promoter. In addition, the plasmid includes a constitutive promoter that drives an immunity protein linked to a fluorescent reporter protein. A second identical constitutive promoter drives a second immunity protein (exemplary plasmid maps are shown in fig. 11 and 21). Each plasmid construct was expressed in MG1655 E.coli. The initial strains, referred to as "strain a" and "strain B", produced colicin E3+ E3+ E7+ sfGFP and colicin E7+ E7+ mCherry, respectively (fig. 1A). To study the kinetics of our engineered strains, a microfluidic device containing rectangular wells of size 100 μm x 80 μm x 1 μm was used. Strain B, designated "disadvantaged" was initially inoculated into a microfluidic device and allowed to fully colonize the wells. At this time, strain a, which is called "dominant strain", is introduced into the microfluidic device. After introduction of strain a, an exponential increase in the dominant strain and a decay in the disadvantaged strain were observed (fig. 1B). These experiments indicate that colicin can be used to control population dynamics. However, one possible outcome of this experiment may be that the "disadvantaged strain" is mutated relative to the dominant strain due to prolonged exposure to low concentrations of colicin.

To solve this problem, it was concluded that simultaneous release of colicin could simultaneously release more lethal concentrations of colicin, while also limiting the time of exposure to low concentrations of colicin. To test this hypothesis, a p15A starting plasmid was constructed in which the original Col E1 lytic protein was removed and replaced with X174E lytic protein under the control of the luxI promoter (fig. 1C). The second plasmid was derived from a synthetic shake strain reported elsewhere (Danino et al, A synchronized quorum of genetic clocks., Nature, 463 (7279): 326-330, 2010), comprising luxI and luxR driven by a native bidirectional promoter on the ColE1 starting plasmid ("activator plasmid"). In addition, the second lux promoter drives the production of a fluorescent reporter protein. In this dual plasmid architecture, a positive feedback loop is established to drive population splitting using the lux population induction system of Vibrio fischeri (Alivbrio fischeri). Briefly, LuxI catalyzes the production of the diffusible quorum sensing molecule, N-Acyl Homoserine Lactone (AHL). This molecule binds to constitutively produced luxR transcription factor to form a complex that serves as a transcriptional activator for the luxI promoter. When both populations are at the lowest level, it is expected that the different strains will lyse simultaneously, resulting in the simultaneous release of colicin (FIG. 1D). To verify this idea, E.coli containing both plasmids was cultured in the microfluidic device described previously. The dominant and disadvantaged strains were loaded at a ratio of 1:5, respectively, and co-cultured for 600 minutes. It was observed that both strains lysed simultaneously in each chamber and the strain inoculation rate was significantly improved compared to the strain lacking simultaneous lysis (fig. 1E). This synthetic colicin-based loop indicates that, when combined with the selective advantages of engineering, cleavage-mediated population control is a powerful tool for controlling populations.

Example 2 three Strain System

In the second stage of development, the system was expanded to three strains.

Since the two-strain population control system can only realize the conversion from one strain to another strain, the design is expanded to a three-strain system, and the circulation can be carried out by adopting a 'stone scissor cloth' method. To explore this, a third toxin was identified as colicin V, which acts by disrupting membrane potential. Each strain contained the same two-plasmid architecture described in fig. 1C, but with orthogonal colicin groups. These three strains, now referred to as strain 1, strain 2 and strain 3, contain colicin E3, colicin E7 and colicin V, respectively. In this system, each strain produced one colicin lethal to the previous strain and two immunity proteins (fig. 2A). The first immunity protein protects the current strain from the toxicity of its own colicin production. In addition, the second immunity protein protects the current strain from the toxicity of colicin produced by the previous strain.

Three E.coli strains were engineered, each containing a unique toxin and antitoxin system (TA modules). In monoclonal populations, the TA module contributes to plasmid stabilization, as mutation or loss of the antitoxin can lead to cell death due to the presence of extracellular toxins produced by healthy bacteria or by killing nascent plasmid-free cells through prolonged persistence of toxin-producing mRNA (see k. gerdes, Nature Biotechnology 6,1402 (1988)). External control of existing population replacement was achieved by extending the mechanism of TA modules to a multi-strain ecology, each TA module conferring additional capacity to its host strain to kill any other strain without the corresponding antitoxin (fig. 12A). Each strain was killed by subsequent strains in one cycle by adding a second antitoxin to each strain, creating a synthetic "stone scissor cloth" (RPS) ecology (fig. 12B). The RPS system may be coupled to any loop of interest to increase stability. In such a combined system, the loop in which the mutation occurs in one strain can be replaced by introducing a new batch of the next RPS-carrying strain. Thus, rather than attempting to "beat darwinian", an engineering strategy is developed that enables external control over the evolution and composition of ecosystems.

Since the multi-strain competition kinetics are usually determined by differences in growth rates, the kinetics of the three strains were first determined separately. The growth rates of 3 lytic strains were compared to the growth rate of wild-type MG1655 E.coli by fluorescence microtiter plate reader absorbance assays. At the occurrence of a cleavage eventPreviously, it was found that the addition of the engineered loop had little effect on growth compared to the wild type control. The OD was recorded at lysis of the three strains₆₀₀Threshold value (fig. 2B).

Next, the cleaved OD was explored in a microfluidic device₆₀₀The effect of the difference in (b) on the lysis period. Each strain was loaded into a microfluidic device and imaged every 6 minutes. Although OD₆₀₀The lysis threshold was different and the lysis period was relatively consistent between the three strains (fig. 2C).

Without wishing to be bound by theory, it is hypothesized that this may be due to the lower survival rate of the cells of strain 1 after lysis compared to the surviving cells of strains 2 and 3. The lysis kinetics of the three strains are phenotypically different. The lysis event of strain 1 resulted in a more explosive lysis event, which left a smaller share of viable cells and drained more lysate from the trap. Alternatively, strain 3 had a very mild lytic event, resulting in a higher share of viable cells and more cell lysate accumulated in the microfluidic well (fig. 7).

In any circumstance, once engineered cells are mutated, they cannot be restored to the unmutated state. They must be removed and replaced by healthy cells. Such a "system restart" can disrupt dynamic loop functionality in the environment. Theoretically, a multi-strain RPS ecosystem should be able to remove mutated or mutable cells without disrupting the loop dynamics of interest. After verifying the extended functionality of the TA module in the RPS system, the utility of the stable RPS element of the genetic circuit with highly selective pressure was tested. The population-driven Synchronous Lytic Circuit (SLC) was selected and the microbial treatment of SLC in vivo, such as tumor administration, was studied (see, e.g., M.O.Din, et al, Nature 536,81 (2016); T.Danino, O.Mondrag Lo-Palomino, L.Tsiming, J.Hasty, Nature 463,326 (2010)). In the SLC loop, a quorum sensing molecule (AHL) accumulates gradually in the growth environment, in a ratio to population density. When the population reaches a density threshold, simultaneous lysis eliminates about 90% of the bacteria, leaving about 10% for re-inoculation of the population for growth. The resulting cell population dynamics is a cycle of cell growth and simultaneous lysis. However, when used in an in vivo environment where selective media cannot be used, plasmid loss or mutation is expected to result in loss of function over a long period of time.

It was investigated whether successful stabilization of the plasmid in the RPS system would lead to strain takeover without interrupting any oscillation kinetics (fig. 13A). The SLC and TA modules were integrated into a two-plasmid system, where one plasmid contained the lytic gene E, TA module from phage Φ X174(X174E), an immune protein, and an activator reporter plasmid (fig. 13B). To investigate how the different kinetics of the strains affect the interactions between them, each strain pair was tested in a microfluidic device. Due to the difference in lysis thresholds, strain 1 is expected to lyse before strains 2 and 3, causing problems with strain takeover. A three strain RPS system was developed in which each strain contained a different toxin-antitoxin pair and a second antitoxin to the other strain (fig. 12C). To achieve this, used colicin, a naturally occurring toxin-antitoxin system, is used, is lethal to certain strains of E.coli and is a potent antagonist in E.coli populations in vivo (FIGS. 16A-D) (see, e.g., B.C.Kirkup, M.A.Riley, Nature 428,412(2004), A.E.Boyer, P.C.Tai, Journal of bacteriology 180,1662 (1998); E.Cascales, et al Microbiology and molecular biology reviews 71,158 (2007); M.M.Zambrano, D.A.Siege, M.Almirron, A.Tormo, R.Kolter, Science 259,1757 (1993)).

To test this, each strain pair was co-cultured in a microfluidic device at an initial inoculation ratio of 1:5 (preponderance versus susceptibility). Each of the three strains was monitored by fluorescent reporter proteins sfGFP, CFP and mKate 2. The generation of the fluorescent reporter is driven by the luxI promoter. Strain 3 contains plasmids producing the E7 and E7 immunity proteins and the E V immunity protein. Strain 2 contains plasmids producing the E3 and E3 immunity proteins and the E7 immunity protein. Strain 1 contains plasmids producing colicin V and V immunity proteins and E3 immunity protein. Thus, each pair of strains has a "dominant strain" that is immune to the toxin produced by the other strain, and a "susceptible strain" that is sensitive to the toxin produced by the other strain. Thus, the reporter protein is only produced after the initiation of a positive feedback loop based on the AHL population.

Since the replacement in microbial co-cultures was generally determined by differences in growth rate, the growth rate of each RPS strain was measured (fig. 12D). Co-culture was performed at a ratio of dominant to susceptible strains of 1:2 to determine the efficacy of the engineered colicin killing circuit in a microfluidic device. In each case, a rapid increase of the dominant strain and a decline of the susceptible strain were observed, confirming that colicin produced by the dominant strain was effective against the susceptible strain, but vice versa (fig. 12E, 12F).

Three strains were generated using this architecture, which showed simultaneous cycles of simultaneous lysis and constant RPS competition (fig. 13C). Each RPS lytic strain was characterized separately (FIGS. 17A-D), and each pair was verified in a microfluidic device at initial inoculation ratios of 1:1 and 1:5 for the dominant versus the susceptible. It was observed that the function of the simultaneous lysis loop was unaffected, except for successful strain displacement, as all strains lysed simultaneously in each chamber. At a 1:1 ratio of dominant to susceptible bacteria, take-over occurred for 100% of the cultures (n-35) per strain, and a single lysis event was sufficient to complete the strain take-over (fig. 18A). For the three pairings at a ratio of 1:5 (strains 1 and 3, 3 and 2, 2 and 1), dominant strain takeover occurred in 100%, 100% and 92% of the cultures (n ═ 396), respectively (fig. 2D, fig. 18B). For co-cultures consisting of strain 1 and strain 2, a small share (< 8%) of strain 2 was able to compete for strain 1 to colonize in the well. In all cases, the function of the SLC loop was not interrupted during strain switching (fig. 13D-13F).

Without wishing to be bound by theory, it is hypothesized that this may be caused by the lower lysis threshold of strain 1. Strain 1 began lysis prior to strain 2. Therefore, the relative concentration of the released colicin with respect to the target strain 2 was low. Furthermore, although the two strains were initially loaded with a 1:5 ratio of superiority to inferiority, in most cases the ratios observed at the first cleavage event were different.

To further investigate this problem, we performed a microfluidic experiment with two strains loaded at a 1:1 ratio. In most cases, the dominant strain has completely taken over the microfluidic trap before the first lysis event occurs. In a few cases, both strains were still present in the trap. However, the dominant strain has a larger population size, resulting in a single lytic event sufficient to take over the trap (see, e.g., FIGS. 2E-2G, FIGS. 13D-13F).

Microscopic examination (60-fold magnification) at single cell resolution visually confirmed that the vulnerable cells had begun to die before the simultaneous lysis. Without being bound by theory, this may indicate that there is some degree of "leaky expression" of the cleaved protein during the exponential growth phase, resulting in the release of small amounts of colicin.

Example 3 population dynamics

While co-cultivation of dominant-susceptible strain pairs enables constant strain replacement, ecosystems consisting of three antagonistic strains may introduce newly emerging characteristics when cultured simultaneously. A mathematical model describing the dynamics of the switching population was developed and reduced to a discrete time plot that allows analytical prediction of the switching frequency and the interval time (see example 5). The results show that population dominance cycling between the three strains is a emergent feature of the system when all three strains are present simultaneously. Strains initially starting at higher concentrations then predominate in the wells, assuming the system does not allow the introduction of fresh cells (fig. 19). Alternatively, under small constant feeding conditions for each strain, the trajectory of the three-strain system converged to a stable limit cycle regardless of the initial ratio between the three strains (fig. 3E, fig. 13G).

To test this emergent property, all three strains were loaded simultaneously into one microfluidic device. The theoretical robustness of the system was tested using the same experimental setup described above. The only difference in the protocol is the use of high flow rates after cell loading in the microfluidic device. This process is to flush most of the cells out of the wells, leaving only about 1 to 10 cells in each well. Using this method, a broad relative ratio between the three strains was obtained in different wells, allowing testing of widely distributed initial concentrations.

Four separate microfluidic experiments were analyzed for a total of 1582 microfluidic wells. Three different kinetics were observed. Four independent microfluidic experiments were analyzed, for a total of 1582 individual microfluidic culture zones, and the system was observed to exhibit cycling behavior between three strains, as strain 2 through strain 1 through strain 3 (fig. 20A-20B). The kinetics of about 75% of the traps consisted of one strain competing against the other before the first lytic event occurred. A single strain dominates the trap throughout the experiment. In the second case (-20%) the trap was cycled between two strains, e.g., strain 1 through strain 2. In the third case (. about.5%) the trap was cycled between three strains, e.g.Strain 1 to Strain 2 to Strain 3. By summarizing all transition events in four experiments, experimental results were observed to fit well with theoretical trajectories (fig. 3F). As predicted by the model, the three strains compete with each other until two strains are eliminated and one survived (fig. 3F, fig. 13H).

Under ideal experimental conditions, a constant supply of the next strain is provided in the cycle. However, due to experimental limitations, strain cycling relies on the cells to wash into the trap from upstream traps and channels. It was confirmed that the strains were circulating sequentially under variable conditions including all 3 strains and all 3 colicins. However, due to the geometry of the wells, as the duration of the experiment increases, the accumulation of cell lysate in the microfluidic wells prevents new cells from being washed out upstream, reducing the capacity of the strain to circulate continuously. Due to the limitations of these microfluidic experiments, it is not possible to show a complete cycle of the system in one run. However, several examples are presented in which the strain is cycled from strain 2 to strain 1, then to strain 3, and other protocols (fig. 3D-3E, fig. 13G). However, by summarizing the data from the four experiments and 1600 microfluidic wells, it was demonstrated that changes in the initial conditions of the system (i.e., the initial concentration of each strain) did not prevent the oscillations between the three strain systems from stabilizing, one strain cycling sequentially at a time (fig. 3C, fig. 19A-19B, fig. 9). This demonstrates the robustness of the system, since changes in initial conditions do not perturb the steady state of the system. For in vivo applications, this means that achieving the desired strain cycle is not too sensitive to initial conditions (e.g., loading concentration, loading time, etc.).

The consistent convergence of the RPS ecosystem with expected behavior supports the feasibility of the engineered ecosystem to exhibit predictable and accurate dynamics. The potential of synthetic microbial communities for new biotechnological applications has long been known (see, e.g., B.Kerr, M.A.Riley, M.W.Feldman, B.J.Bohannan, Nature 418,171 (2002); L.R.Lynd, et al, Nature biotechnology 26,169 (2008); T.Gro. beta. kopf, O.S.Soyer, Current opinion in microbiology 18,72 (2014); K.ZHou, K.Qiao, S.Edgar, G.Stephanopoulos, Nature biotechnology 33,377 (2015)). Engineered ecosystems exhibit complex functions and are difficult to engineer into a single population. (see, e.g., J.Shong, M.R.J.Diaz, C.H.Collins, Current Opinion in Biotechnology 23,798 (2012); K.Brenner, L.you, F.H.Arnold, Trends in Biotechnology 26,483 (2008)).

Example 4 plasmid stability

The RPS synthetic ecosystem strategy allows for external control of ecosystem evolution and composition, by manual input, to replace unwanted strains without disrupting loop function. To demonstrate this concept and to extend the functional stability of SLCs using the RPS system, we attempted dynamic expression without a selective antibiotic (kanamycin). In this case, selective pressure against the SLC activator plasmid should result in rapid loss of function.

Two cases were investigated (fig. 14A). In case 1, each strain was cultured separately in LB medium lacking kanamycin. In the absence of selective antibiotics, loss of circuit function is common and results in loss of fluorescence expression and simultaneous lysis kinetics. The time elapsed before the loss of the synchronous lysis (n-16) was recorded and 80% to 90% of the plasmid loss was found to occur within 32 hours, after which the strain growth was not controlled (fig. 14B, 14C, l.o.f. for "loss of function"). Under the 2 nd protocol, the experiment started with co-cultivation of strain 2 and strain 1, and strain 1 was allowed to take over the trap. After 12 hours, strain 3 was added to replace strain 1, and after 30 hours strain 2 was reintroduced to replace strain 3. Thus, starting from strain 2, a complete cycle is completed in a manner that may be maintained indefinitely. By manually adding subsequent strains before the previous strain mutates, the duration of loop stability can be extended without disrupting loop function (fig. 14D).

To further explore the effect of the RPS system on mutable genetic circuits, a batch passage experiment was performed to compare the two protocols (fig. 15A). Under the first protocol, a medium containing antibiotics was inoculated with strain 1 and the culture was passaged to fresh growth medium every 12 hours. Under the second protocol, the cultures were also inoculated with strain 1 and passaged every 12 hours, but the next strain in the RPS cycle was also added every 3 passages. It was observed that in the first scenario, the mutation started from passage 4 and could no longer be recovered once loss of sync has occurred. However, under the second protocol, it was observed that even if the simultaneous lysis was lost in the previous passage, there was a delay in the loss of simultaneous lysis and lysis function would be restored (fig. 15B). Sanger sequencing of the 1kb pair region containing the X174E cleavage gene and the Lux cassette showed that the RPS strategy reduced the occurrence of mutations within the same duration. In this way, the RPS system's ability to prevent loss of loop function, whether by mutation or plasmid loss, was demonstrated by shifting the challenge of eliminating a single mutant cell to eliminating the entire population.

As an extensible and modular platform to ensure genetic stability, the RPS system provides an additional layer of control that allows it to maintain plasmid stability in conjunction with other traditional strategies. This approach can allow synthetic biologists to engineer systems that can be maintained for long periods of time without selective antibiotics, affecting applications from therapy to bioremediation, production, and sensing technologies. These applications will require the development of new RPS strains with different natural or synthetic toxin-antitoxin systems, since the prevalence of colicin resistance may occur relatively rapidly depending on environmental conditions. In a broader scope, the possibility of using dynamic synthetic ecosystems is created by the programmed manipulation and interaction of independent sub-populations, using engineered "synthetic ecology" to control the stability of genetic constructs.

Example 3 model

These observations were integrated into a computational model to visually display the behavior of the three strain lysis-based system under various parameters. Assuming that each strain could be completely killed by another, it was found that each of the three strains cycled once and the strain that initially started at a higher concentration would dominate the well (fig. 3A). All three strains were loaded simultaneously into one microfluidic device. All three strains were grown to the same approximate OD and then mixed together. The cell mixture is then loaded into a microfluidic device. The system was able to cycle between three strains, e.g., strain 2 through strain 1 through strain 3 (fig. 3B-C). No recycling of the strain back to the first strain to complete the cycle was observed. Due to the lethal effect of one strain on another, these three strains can compete until only one champion remains.

It is hypothesized that to achieve persistent transitions in the model, the addition of a small epsilon (epsilon) representing a constant inoculum of cells helps to avoid complete elimination of either strain (fig. 3D). The epsilon term represents the continuous addition of the next strain in the system to provide a constant supply of each cell type. Furthermore, the trajectory of the three strain system converged to a stable limit cycle that was robust to a wide range of initial conditions and system parameters (fig. 8).

The three bacterial strains have densities p, q, r, which grow exponentially at the same nominal growth rate in the absence of colicin, and can be scaled in ratio without loss of generality. These three strains produce three types of colicin. The concentration of each of these colicins in the extracellular medium was C_p，C_q，C_r. The role of these colicins is that they slow down each other's growth in a cyclic manner: c_pSlow the growth of q, so that q is (1+ C)_p)^-1Rate of increase of C_qAlso slow down r, C_rP is slowed down. In addition, all strains produced the quorum sensing molecule AHL, the extracellular concentration of AHL being denoted as a.Colicin produced by the cells remains associated with the corresponding cells and does not affect other strains until released into the extracellular medium during the lysis event. If A is greater than or equal to A _cAll strains are lysed, during which most cells are lysed, but a small fraction of δ survives. AHL in the extracellular space is constantly diluted at a rate γ A, while colicin is at a rate γ_cAnd (6) diluting.

Assume time t immediately after cleavage event i_iAll 7 concentration values are p_i,q_i,r₁, A_i. Note that assume A_i＝A_c(lysis events are short and there is no chance of change in AHL concentration). The subsequent evolution of these kinetic variables is governed by the following equation:

after cleavage event i, the concentrations of p, q, r were small and the concentration of AHL began to drop due to dilution. However, as the total density of bacteria, p + q + i, increased sufficiently, the concentration of AHL began to rise back up. When A is at time t_i+1Again reach A_cWhen this happens, a new cleavage event occurs. At this time, are respectively equal to P_i+1、Q_i+1、R_i+1The concentration of the three strains immediately drops by a fraction delta,

|p_i+1＝δP_i+1+∈ (8)

q_i+1＝δQ_i+1+∈ (9)

r_i+1＝δR_i+1+∈ (10)

[ note: a small epsilon was added to avoid complete elimination of each strain. Seemingly trivial but crucial persistent switching seems to be left alone ]. At the same time, colicin is released from the lysed cells into the extracellular space and added to the colicin previously present therein:

here, theIs the concentration of strain p immediately before i +1 cleavage events, similar to Q and R. The parameter beta represents the rate at which colicin is released by the bacteria during lysis (assuming that the release rates are the same for all three strains).

The simulations of equations (1) - (13) show the kinetics of robust transitions in dominant strain density over a wide range of parameter values (fig. 24A, 24B, 25A, 25B, 26A and 26B).

Analysis of

The Escilin kinetic equations between cleavage events are linear, so they can be simply integrated,

now, since the change in colicin concentration with time is known, the equation for the strain concentration can be integrated:

finally, the concentration of AHL between lysis events was calculated as:

determining the time of the next cracking event from the transcendental equation

From now on, it is assumed that AHL kinetics are much faster than population kinetics (i.e. γ)_A>>1). AHL concentration then tracks the total cell mass in the chamber, simplifying expression of a,

thus the next cleavage event condition becomes

P_i+Q_i+R_i＝γ_AA_cα^-1 (22)

If the colicin degradation rate is sufficiently fast, then at the end of each lysis cycle they have time to degrade to negligible minute values, as shown in FIGS. 25A, 25B, 26A and 26B, thenThe value is independent of the colicin concentration in the previous lysis cycle and is according to equations (23) - (25) and P_i、Q_i、R_iIn proportion:

under the circumstancesOne cleavage event t ═ t_iSubstituting these expressions and (8) - (10) into the expressions (15) - (17) at +1 time results in the following implicit mapping

Wherein, the symbol T_i＝t_i+1-t_iFor the time interval between the i-th and (i +1) -th cleavage events. The mapping is still implicit, since the Ti value is still uncertain. If δ < 1, i.e. the cell density increases significantly between lysis events, the expression in brackets can be simplified by decreasing 1:

t can now be calculated by adding these tree equations and using equation (22)_i：

Finally, P is realized_i，Q_i，R_iIs shown as followsThree-dimensional mapping:

(36)

wherein:

the intervals between cleavage events from equation (32) are as follows:

the immobile point bifurcates from Hopf.

Mapping the immobilization points of (33) - (33), wherein all three strains are equal, see P_i＝Q_i＝R_i＝P₀＝γ_AA_cAnd/3 alpha. In this protocol, the time between cleavage events is as follows:

(45)

in order to investigate the stability of this stationary point, the map in the vicinity thereof was linearized,

wherein X₀＝(δP₀+ε)(1+βP₀)^-1/γcAnd is

Substituting these expressions results in the following linear mapping:

wherein:

the stability of the stationary point is determined by the eigenvalues of the following feature matrix:

the matrix has three eigenvalues: lambda [ alpha ]₁＝0；λ_2；33A +3B/2+ i3 sqrt (3) B/2. The first characteristic value is 0, corresponding to super stability. The second and third eigenvalues are complex eigenvalues and may correspond to an exponentially growing solution if their absolute value | λ 2, 3| is greater than 1, sqrt ((3A +3B/2) ²+27B²/4) > 1, or

A²+B²+AB＞1/9

This is the condition for the Hopf bifurcation in this model. It can be easily seen that for small A_cThe left hand side is small, but it follows A_cMonotonically increasing to asymptotic values greater than 1/9, so that Hopf bifurcation always occurs at some finite A_cHere (see fig. 8A). At the Hopf bifurcation, where | λ_2，31, the number of cracking events per conversion cycle (1/3 of cycle) is

Fig. 8B and 8C show graphs of numbers of cleavage pulses and bifurcations for the map and the underlying intact model.

Strong conversion scheme

Away from the Hopf bifurcation point, when one strain dominates the dynamics over many lysis cycles, then switches rapidly to the next strain dominance, the transition becomes strongly nonlinear, and so on (fig. 25, 26). To determine the kinetics of the protocol, the time intervals between cleavage events and the duration of the switching cycles were calculated.

The cleavage interval is varied within one switching cycle. When a strain is strongly dominant, they are minimal in the middle of the cycle; when the two strains are nearly equal, they are slightly longer between cycles.

Minimum cleavage Interval

During most of each conversion cycle, one of the strains grew to a high concentration at which lysis occurred, while the other two strains remained at a smaller concentration (assuming ε < > _γAA/alpha). In this case, the mapping is almost one-dimensional and small (without loss of generality, p is chosen as the dominant strain, assuming Q_i＝R_i0 is:

P_i＝γ_AA_cα^-1＝P_M， (56)

[ suppose we assume ε < δ γ_AA/alpha. The intervals between cleavage events at this stage are as expected

Maximum cleavage interval. The maximum lysis interval occurs during the transition from dominance of one strain to the next. During this transition, the two concentrations become equal briefly at a certain i ═ k +1 (assuming for clarity, P is equal_k+1＝Q_k+1，R_k+10). It is easy to see equation P_k+1＝Q_k+1Provided that

(small epsilon is ignored here). For sufficiently sharp transitions, the webValue P_kIs still close to P_M＝_γAA_cA,/α. This allows solving for R_kIt can then be used to find T_k. If beta R_k> 1 (to be verified later) 1 can be ignored, the equation can be solved explicitly,

from this, the expression is for γ_c1 is beta gamma₄A_cα > 1. The maximum cleavage interval is then explicitly calculated:

mean cleavage Interval T_aBetween T_mAnd T_MWhile gradually approaching T as the transition interval becomes longer_m。

The transition interval duration. To estimate the duration of the switching cycle for which a strain predominates, it was observed to consist of two subintervals. In the first subinterval, one of the strains (with specificity P) predominates, remaining at P before the lytic event occurs _MNearby, while the other two strains (q and r) were smaller. One of the strains (q) was very small (O (. epsilon.)) throughout the subinterval, since its growth was strongly inhibited by large P, while the other strain (r) was uninhibited, growing steadily from a small initial level to O (P)_M) Eventually reaches the same value as P_MA comparable amplitude. Once this occurs, the second subinterval begins, during which the amplitude of the first strain (p) rapidly decreases to a small (O (epsilon)), as its growth is now inhibited by a large r. To obtain the duration of the first subinterval, consider R_iIs given by the mapping of_i≈0,P_i≈P_M

According to the equation (32),

using the expression in equation (31), the following 1D mapping is obtained

To obtain the duration of the first subinterval, R is determined_iFrom R₀Starts to reach P when-0_MAn order of magnitude of the number of iterations. This sub-interval may itself be divided into two parts. When beta R_i<<1, the second bracket is expanded to obtain the following quadratic mapping

Or introducing new variables

Wherein a ═ epsilon beta/delta gamma_cInitial conditionsFor small a<<1, to achieveIs large, by taking successive limits and applying the following equationsToIntegration is performed to obtain:

wherein for small a is obtained

Of course, the last few iterations violate the condition β R _i<<1, but this expression still evaluates well to reach R_n＝β^-1The number of iterations required. Next, R is estimated_iFrom Rn ═ beta^-1To P_MAdditional iterations of (a). Due to the assumption of beta>>ε/δ at the R_iInsofar, the latter is omitted from the mapping (63) and reduced to

WhereinInitial conditionsThe first few iterations of the mapping are as follows: and the like. For gamma_cO (1), the sequence grows rapidly (super-exponential) with i, so after i n +2, 1 is ignored in parentheses in equation (68), simply written as

Thus in N₁In the iteration process, the data of the data acquisition system,

equating the expression to PM and recalling the expression of N to obtain the following N₁The approximate formula of (a) is,

during the second sub-interval, the magnitude of Ri has approached P_MThus, the growth of the P strain is strongly inhibited. This means that in each cleavage event it decreases by a factor delta, P_i+1＝δP_iThis immediately produces P_iAn estimate of the number of iterations required to reach a "background" level O (epsilon),

thus, the total number of cleavage intervals within a single conversion cycle is given by

Figures 8D-8G show the intervals between cleavage events (min, max, and mean) and the number of cleavage events within each conversion interval, as a function of some model parameters, obtained from the exact model, 3D mapping, and analysis approximation. Surprisingly, the analytical approximation appears to be more accurate than the three-dimensional mapping results.

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Other embodiments

Embodiment 1 is a method, comprising:

culturing a first bacterial strain in a growth environment for a first period of time;

adding a second bacterial strain to the growth environment and culturing the second bacterial strain for a second period of time;

adding a third bacterial strain to the growth environment and culturing the third bacterial strain for a third period of time;

Wherein each of the first, second and third strains comprises a toxin system;

optionally wherein each of the first, second and third strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule.

Embodiment 2 is the method of embodiment 1, wherein the quorum sensing molecule is different in each of the first, second, and third strains.

Embodiment 3 is the method of embodiment 2, wherein the quorum sensing molecule of each of the first, second, and third strains has no or substantially no effect on the activatable promoter of the lytic gene of the other strain.

Embodiment 4 is the method of embodiment 1, wherein the quorum sensing molecule is the same in each of the first, second, and third strains.

Embodiment 5 is the method of any one of embodiments 1-4, further comprising culturing or co-culturing one or more additional bacterial strains in a growth environment.

Embodiment 6 is the method of embodiment 5, wherein each of the one or more additional bacterial strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule.

Embodiment 7 is the method of embodiment 6, wherein the quorum sensing molecule is different in each of the first, second, and third and one or more additional bacterial strains.

Embodiment 8 is the method of embodiment 7, wherein the quorum sensing molecule of each of the first, second, and third strains and the one or more additional bacterial strains each has no effect or substantially no effect on an activatable promoter of a lytic gene of another strain used in the growth environment.

Embodiment 9 is the method of embodiment 6, wherein the quorum sensing molecule is the same in each of the first, second, and third and one or more additional bacterial strains.

Embodiment 10 is the method of any one of embodiments 6-9, wherein each of the one or more additional bacterial strains includes a toxin system.

Embodiment 11 is the method of any one of embodiments 1-10, wherein the toxin system of each of the first, second, third and one or more additional strains may be independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 12 is the method of any one of embodiments 1-11, wherein the lytic plasmid and the activator plasmid of each of the at least first, second, and third bacterial strains are copies of the same plasmid.

Embodiment 13 is the method of any one of embodiments 1-11, wherein the lytic plasmid and the activator plasmid of each of the at least first, second, and third bacterial strains are different plasmids.

Embodiment 14 is the method of any one of embodiments 1-13, wherein at least the first, second, and third bacterial strains have metabolic competition.

Embodiment 15 is the method of any one of embodiments 1-14, wherein at least the first, second, and third bacterial strains are selected from escherichia coli, salmonella typhimurium, or bacterial variants thereof.

Embodiment 16 is the method of any one of embodiments 1-15, wherein each of the at least first, second, and third bacterial strains has no growth advantage over another strain in the growth environment.

Embodiment 17 is the method of any one of embodiments 1-16, wherein, in each of the at least first, second, and third bacterial strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene.

Embodiment 18 is the method of any one of embodiments 1-17, wherein the lytic gene in each of at least the first, second, and third bacterial strains is E from bacteriophage Φ X174.

Embodiment 19 is the method of any one of embodiments 1-18, wherein the activatable promoter in each of at least the first, second and third bacterial strains is a LuxR-AHL activatable luxI promoter and the activator gene is luxI.

Embodiment 20 is the method of embodiment 17, wherein the at least one reporter gene is selected from a gene encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), or variants thereof.

Embodiment 21 is the method of embodiment 17, wherein the degradation tag is an ssrA-LAA degradation tag.

Embodiment 22 is the method of any one of embodiments 1-21, wherein at least one of the plasmids is integrated into the genome of at least one of the first, second, and third bacterial strains.

Embodiment 23 is the method of any one of embodiments 1-22, wherein the culturing is performed in a microfluidic device.

Embodiment 24 is the method of any one of embodiments 1-22, wherein the culturing is performed in a bioreactor.

Embodiment 25 is the method of any one of embodiments 1-22, wherein the culturing is performed in vivo.

Embodiment 26 is the method of any one of embodiments 1 to 25, wherein each of the first, second, and third time periods ranges from about 12 hours to about 72 hours.

Embodiment 27 is the method of any one of embodiments 1-25, wherein the first, second, and third time periods are each selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours.

Embodiment 28 is the method of any one of embodiments 1-25, wherein the first, second, and third time periods are each selected from 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours.

Embodiment 29 is the method of any one of embodiments 1-25, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 72 hours.

Embodiment 30 is the method of embodiment 29, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 48 hours.

Embodiment 31 is the method of embodiment 29, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 24 hours.

Embodiment 32 is the method of embodiment 29, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 18 hours.

Embodiment 33 is the method of embodiment 29, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 12 hours.

Embodiment 34 is the method of embodiment 29, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 6 hours.

Embodiment 35 is the method of embodiment 29, wherein each of the first, second, and third time periods independently ranges from about 1 hour to about 3 hours.

Embodiment 36 is the method of any one of embodiments 1-35, wherein at least the first, second, and third time periods occur sequentially.

Embodiment 37 is the method of any one of embodiments 1 to 36, wherein the first and second, second and third, or first and third time periods partially or completely overlap.

Embodiment 38 is the method of any one of embodiments 1-37, wherein one or more of the first, second, and third bacterial strains encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

Embodiment 39 is the method of embodiment 38, wherein the promoter is an activatable promoter.

Embodiment 40 is the method of embodiment 39, wherein the promoter is activated by the quorum sensing molecule.

Embodiment 41 is the method of embodiment 38, wherein the promoter is a constitutive promoter.

Embodiment 42 is the method of any one of embodiments 38-41, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

Embodiment 43 is the method of embodiment 42, wherein the therapeutic agent is selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

Embodiment 44 is the method of embodiment 43, wherein the inhibitory nucleic acid is an siRNA, shRNA, miRNA, or antisense.

Embodiment 45 is a method, comprising:

providing n bacterial strains including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain;

co-culturing each strain in turn in a growth environment for an independent period of time;

wherein each of the second through nth bacterial strains may have a previous bacterial strain;

Wherein n is at least 3;

wherein each of the n strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein each of the n bacterial strains comprises a toxin system;

Embodiment 46 is the method of embodiment 45, wherein, in each of the n strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene.

Embodiment 47 is the method of any one of embodiments 45-46, wherein the lytic gene in each of the n bacterial strains is E from bacteriophage Φ X174.

Embodiment 48 is the method of any one of embodiments 45-47, wherein the activatable promoter in each of the n bacterial strains is a LuxR-AHL activatable luxI promoter and the activator gene is LuxI.

Embodiment 49 is the method of embodiment 46, wherein the at least one reporter gene is selected from a gene encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), or variants thereof.

Embodiment 50 is the method of embodiment 46, wherein the degradation tag is an ssrA-LAA degradation tag.

Embodiment 51 is the method of any one of embodiments 45-50, wherein at least one of the plasmids is integrated into the genome of at least one of the n bacterial strains.

Embodiment 52 is the method of any one of embodiments 45-51, wherein the quorum sensing molecule is different in each of the n bacterial strains.

Embodiment 53 is the method of embodiment 52, wherein each of the quorum sensing molecules of each of the n strains has no effect or substantially no effect on the activatable promoter of the lytic gene of the other strain.

Embodiment 54 is the method of any one of embodiments 45-51, wherein the quorum sensing molecule is the same in each of the n bacterial strains.

Embodiment 55 is the method of any one of embodiments 45-54, wherein the lytic plasmid and the activator plasmid of each of the n bacterial strains are copies of the same plasmid.

Embodiment 56 is the method of any one of embodiments 45-54, wherein the lytic plasmid and the activator plasmid of each of the n bacterial strains are different plasmids.

Embodiment 57 is a method, comprising:

providing n bacterial strains including at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain;

co-culturing each strain in turn in a growth environment for an independent period of time;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

Wherein the toxin system of the first bacterial strain produces a third toxin/third antitoxin pair and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Embodiment 58 is the method of any one of embodiments 44-57, wherein the n bacterial strains are metabolically competitive.

Embodiment 59 is the method of any one of embodiments 44-58, wherein each of the n bacterial strains is selected from escherichia coli, salmonella typhimurium, or a bacterial variant thereof.

Embodiment 60 is the method of any one of embodiments 44-59, wherein each of the n bacterial strains has no growth advantage over another strain in the growth environment.

Embodiment 61 is the method of any one of embodiments 44-60, wherein the toxin systems of the n bacterial strains are independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 62 is the method of any one of embodiments 44-61, wherein the culturing is performed in a microfluidic device.

Embodiment 63 is the method of any one of embodiments 44-61, wherein the culturing is performed in a bioreactor.

Embodiment 64 is the method of any one of embodiments 44-61, wherein the culturing is performed in vivo.

Embodiment 65 is the method of any one of embodiments 44-64, wherein each separate period of time ranges from about 12 hours to about 72 hours.

Embodiment 66 is the method of any one of embodiments 44-64, wherein each independent time period is selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours.

Embodiment 67 is the method of any one of embodiments 44-64, wherein each independent time period is selected from 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours.

Embodiment 68 is the method of any one of embodiments 44 to 64, wherein each separate time period independently ranges from about 1 hour to about 72 hours.

Embodiment 69 is the method of embodiment 68, wherein each period of time independently ranges from about 1 hour to about 48 hours.

Embodiment 70 is the method of embodiment 68, wherein each period of time independently ranges from about 1 hour to about 24 hours.

Embodiment 71 is the method of embodiment 68, wherein each period of time independently ranges from about 1 hour to about 18 hours.

Embodiment 72 is the method of embodiment 68, wherein each period of time independently ranges from about 1 hour to about 12 hours.

Embodiment 73 is the method of embodiment 68, wherein each period of time independently ranges from about 1 hour to about 6 hours.

Embodiment 74 is the method of embodiment 68, wherein each period of time independently ranges from about 1 hour to about 3 hours.

Embodiment 75 is the method of any one of embodiments 45-74, wherein one or more of the time periods partially or completely overlap.

Embodiment 76 is the method of any one of embodiments 45-75, wherein one or more of the n bacterial strains encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

Embodiment 77 is the method of embodiment 76, wherein the promoter is an activatable promoter.

Embodiment 78 is the method of embodiment 77, wherein the activatable promoter is activated by a quorum sensing molecule.

Embodiment 79 is the method of embodiment 76, wherein the promoter is a constitutive promoter.

Embodiment 80 is the method of any one of embodiments 76-79, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

Embodiment 81 is the method of embodiment 80, wherein the therapeutic agent is selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

Embodiment 82 is the method of embodiment 81, wherein the inhibitory nucleic acid is an siRNA, shRNA, miRNA, or antisense.

Embodiment 83 is the method of any of embodiments 45-82, wherein each of the second through nth bacterial strains does not produce the second toxin of the toxin system of the previous strain, and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain.

Embodiment 84 is a bacterial strain comprising a lytic plasmid and an activator plasmid, wherein the lytic plasmid comprises a lytic gene, an activatable promoter, and an optional reporter gene, and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene, wherein the bacterial strain further comprises a toxin system, wherein the toxin system produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the bacterial strain does not produce the second toxin.

Embodiment 85 is the bacterial strain of embodiment 84, wherein the lytic gene is E from bacteriophage Φ X174.

Embodiment 86 is the bacterial strain of any one of embodiments 84-85, wherein the activatable promoter is a LuxR-AHL activatable luxI promoter and the activator gene is luxI.

Embodiment 87 is the strain of any one of embodiments 84-86, wherein the activator gene encodes a molecule that directly or indirectly activates the activatable promoter.

Embodiment 88 is the bacterial strain of embodiment 87, wherein the molecule that directly or indirectly activates the activatable promoter is a quorum sensing molecule.

Embodiment 89 is the bacterial strain of any one of embodiments 84-88, wherein the lytic gene is operably linked to an activatable promoter.

Embodiment 90 is the bacterial strain of any one of embodiments 84-89, wherein the reporter gene on the lytic plasmid is operably linked to an activatable promoter.

Embodiment 91 is the bacterial strain of any one of embodiments 84-90, wherein the activator plasmid further comprises an activatable promoter.

Embodiment 92 is the bacterial strain of embodiment 91, wherein the activatable promoter of the activator plasmid is a copy of the activatable promoter of the lytic plasmid.

Embodiment 93 is the bacterial strain of any one of embodiments 91-92, wherein the activator gene is operably linked to an activatable promoter of an activator plasmid.

Embodiment 94 is the bacterial strain of any one of embodiments 91-93, wherein the reporter gene on the activator plasmid is operably linked to an activatable promoter of the activator plasmid.

Embodiment 95 is the bacterial strain of any one of embodiments 91-94, wherein the degradation tag is operably linked to an activatable promoter of an activator plasmid.

Embodiment 96 is the bacterial strain of any one of embodiments 84-95, wherein the toxin system is operably linked to an activatable promoter that lyses the plasmid.

Embodiment 97 is the bacterial strain of any one of embodiments 84-96, wherein the degradation signature is an ssrA-LAA degradation signature.

Embodiment 98 is the bacterial strain of any one of embodiments 84-97, wherein the reporter gene of the lytic plasmid, the reporter gene of the activator plasmid, or both are fluorescent proteins.

Embodiment 99 is the bacterial strain of any one of embodiments 84-97, wherein the reporter gene of the lytic plasmid and the reporter gene of the activator plasmid are different genes.

Embodiment 100 is a bacterial strain comprising a toxin system, wherein the toxin system produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the bacterial strain does not produce the second toxin.

Embodiment 101 is the bacterial strain of any one of embodiments 84-100, wherein the bacterial strain encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

Embodiment 102 is the bacterial strain of embodiment 101, wherein the promoter is an activatable promoter.

Embodiment 103 is the bacterial strain of embodiment 102, wherein the activatable promoter is activated by a quorum sensing molecule.

Embodiment 104 is the bacterial strain of embodiment 101, wherein the promoter is a constitutive promoter.

Embodiment 105 is the bacterial strain of any one of embodiments 101-104, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

Embodiment 106 is the bacterial strain of any one of embodiments 84-105, wherein the toxin system is independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 107 is the bacterial strain of any one of embodiments 84-106, wherein the bacterial strain further comprises a nucleic acid encoding a therapeutic agent.

Embodiment 108 is the bacterial strain of embodiment 105 or embodiment 107, wherein the therapeutic agent is selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

Embodiment 109 is the bacterial strain of any one of embodiment 105 or embodiment 107-108, wherein the therapeutic agent is a therapeutic polypeptide.

Embodiment 110 is the bacterial strain of any one of embodiment 105 or embodiment 107-109, wherein the therapeutic agent is cytotoxic or cytostatic to a target cell.

Embodiment 111 is the bacterial strain of embodiment 110, wherein the target cell is a cancer cell or an infected cell.

Embodiment 112 is a pharmaceutical composition comprising any one or more of the bacterial strains of embodiments 84-111.

Embodiment 113 is the pharmaceutical composition of embodiment 112, wherein the pharmaceutical composition is formulated for in situ drug delivery.

Embodiment 114 is a system, comprising:

a first bacterial strain comprising a first lytic plasmid and a first activator plasmid; wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene; the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene; wherein the first bacterial strain further comprises a first toxin system; wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin;

A second bacterial strain comprising a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; wherein the second bacterial strain further comprises a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from a first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and

a third bacterial strain comprising a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, optionally a third degradation tag, and optionally a third reporter gene; wherein the third bacterial strain further comprises a third toxin system; wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Embodiment 115 is a system comprising:

a first bacterial strain comprising a first toxin system; wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin, wherein the first bacterial strain optionally further comprises a first lytic plasmid and a first activator plasmid; wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene; the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene;

a second bacterial strain comprising a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain optionally further comprises a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and

A third bacterial strain comprising a third toxin system producing a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the third bacterial strain optionally further comprises a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, an optional third degradation tag, and an optional third reporter gene.

Embodiment 116 is the system of embodiment 114 or embodiment 115, further comprising one or more additional bacterial strains.

Embodiment 117 is the system of any one of embodiments 114-116, wherein the first toxin system, the second toxin system, and the third toxin system are independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 118 is a system, comprising:

n bacterial strains including at least a first bacterial strain, a second bacterial strain and an nth bacterial strain;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

Wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

Embodiment 119 is a system, comprising:

n bacterial strains including at least a first bacterial strain, a second bacterial strain and an nth bacterial strain;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

Wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein each of the n bacterial strains comprises a toxin system;

Embodiment 120 is the system of embodiments 118 or 119, wherein n is 3.

Embodiment 121 is the system of embodiments 118 or 119, wherein n is 4, 5, 6, 7, 8, 9, or 10.

Embodiment 122 is the system of any one of embodiments 118-121, wherein each of the toxin systems is independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 123 is the system of any one of embodiments 118-122, wherein each of the second through nth bacterial strains does not produce the second toxin of the toxin system of the previous strain and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain.

Embodiment 124 is a kit comprising:

a first pharmaceutical composition comprising a first bacterial strain comprising a first lytic plasmid and a first activator plasmid; wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene; the first activator plasmid comprises a first activator gene, a first degradation tag, and optionally a first reporter gene; wherein the first bacterial strain further comprises a first toxin system; wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin;

a second pharmaceutical composition comprising a second bacterial strain comprising a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, a second degradation tag, and optionally a second reporter gene; wherein the second bacterial strain further comprises a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from a first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and

A third pharmaceutical composition comprising a third bacterial strain comprising a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, a third degradation tag, and optionally a third reporter gene; wherein the third bacterial strain further comprises a third toxin system; wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Embodiment 125 is a kit comprising:

a first pharmaceutical composition comprising a first bacterial strain comprising a first toxin system; wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin, wherein the first bacterial strain optionally further comprises a first lytic plasmid and a first activator plasmid; wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene; the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene;

A second pharmaceutical composition comprising a second bacterial strain comprising a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain optionally further comprises a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and

a third pharmaceutical composition comprising a third bacterial strain comprising a third toxin system that produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the third bacterial strain optionally further comprises a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, an optional third degradation tag, and an optional third reporter gene.

Embodiment 126 is the kit of any one of embodiments 124-125, wherein the first toxin system, the second toxin system, and the third toxin system are independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 127 is the kit of any one of embodiments 124-125, further comprising one or more additional bacterial strains in the first, second, third and/or one or more additional pharmaceutical compositions.

Embodiment 128 is a kit comprising:

n bacterial strains including at least a first bacterial strain, a second bacterial strain and an nth bacterial strain;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

Embodiment 129 is a kit comprising:

n bacterial strains including at least a first bacterial strain, a second bacterial strain and an nth bacterial strain;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein the lytic plasmid of each of the n bacterial strains comprises a toxin system;

wherein each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain;

wherein the toxin system of the lysed plasmid of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Embodiment 130 is the kit of embodiment 128 or 129, wherein n is 3.

Embodiment 131 is the kit of embodiment 128 or 129, wherein n is 4, 5, 6, 7, 8, 9, or 10.

Embodiment 132 is a drug delivery system comprising the system of embodiment 114-.

Embodiment 133 is a periodic drug delivery system comprising the system of embodiment 114-.

Embodiment 134 is a method of treating a disease in a subject in need thereof, comprising:

administering to the subject a therapeutically effective amount of each of:

Embodiment 135 is a method of treating a disease in a subject in need thereof, comprising:

administering to the subject a therapeutically effective amount of each of:

a first drug comprising a first bacterial strain comprising a first lytic plasmid and a first activator plasmid; wherein the first lytic plasmid comprises a first lytic gene, a first activatable promoter, and optionally a first reporter gene; the first activator plasmid comprises a first activator gene, optionally a first degradation tag, and optionally a first reporter gene; wherein the first bacterial strain further comprises a first toxin system; wherein the first toxin system produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair, wherein the first bacterial strain does not produce the third toxin;

a second drug comprising a second bacterial strain comprising a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, optionally a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, a second degradation tag, and optionally a second reporter gene; wherein the second bacterial strain further comprises a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from a first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin; and

A third medicament comprising a third bacterial strain comprising a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, optionally a third degradation tag, and optionally a third reporter gene; wherein the third bacterial strain further comprises a third toxin system; wherein the third toxin system produces a third toxin/third antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin.

Embodiment 136 is a method of treating a disease in a subject in need thereof, comprising:

administering to the subject a therapeutically effective amount of each of:

A second bacterial strain comprising a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain optionally further comprises a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and

a third bacterial strain comprising a third toxin system producing a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the third bacterial strain optionally further comprises a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, an optional third degradation tag, and an optional third reporter gene.

Embodiment 137 is a method of treating a disease in a subject in need thereof, comprising:

administering to the subject a therapeutically effective amount of each of:

a second pharmaceutical composition comprising a second bacterial strain comprising a second toxin system; wherein the second toxin system produces a second toxin/second antitoxin pair and a first antitoxin from the first toxin/first antitoxin pair, wherein the second bacterial strain does not produce the first toxin, wherein the second bacterial strain optionally further comprises a second lytic plasmid and a second activator plasmid; wherein the second lytic plasmid comprises a second lytic gene, a second activatable promoter, and optionally a second reporter gene; the second activator plasmid comprises a second activator gene, optionally a second degradation tag, and optionally a second reporter gene; and

A third pharmaceutical composition comprising a third bacterial strain comprising a third toxin system that produces a third toxin/third antitoxin pair and a second antitoxin from the second toxin/second antitoxin pair, wherein the third bacterial strain does not produce the second toxin, wherein the third bacterial strain optionally further comprises a third lytic plasmid and a third activator plasmid; wherein the third lytic plasmid comprises a third lytic gene, a third activatable promoter, and optionally a third reporter gene; the third activator plasmid comprises a third activator gene, an optional third degradation tag, and an optional third reporter gene.

Embodiment 138 is the method of any one of embodiments 134-137, wherein administering comprises sequentially administering to the subject each of the first, second, and third bacterial strains or the first, second, and third pharmaceutical compositions.

Embodiment 139 is the method of embodiment 138, wherein administering comprises administering each of the first, second, and third pharmaceutical compositions simultaneously, and wherein each of the first, second, and third pharmaceutical compositions has a different release profile that releases the first, second, and third bacterial strains.

Embodiment 140 is the method of any one of embodiments 134-139, wherein each of the first, second, and third bacterial strains expresses a different therapeutic agent.

Embodiment 141 is a method of treating a disease in a subject in need thereof, comprising:

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

Embodiment 142 is a method of treating a disease in a subject in need thereof, comprising:

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein the lytic plasmid of each of the n bacterial strains comprises a toxin system;

Wherein the toxin system of the lysed plasmid of the first bacterial strain produces a third toxin/third antitoxin pair, and a fourth antitoxin from a fourth toxin/antitoxin pair produced by the nth bacterial strain, wherein the third toxin is effective against the nth bacterial strain.

Embodiment 143 is the method of embodiment 141 or 142, wherein n is 3.

Embodiment 144 is the method of embodiment 141 or 142, wherein n is 4, 5, 6, 7, 8, 9, or 10.

Embodiment 145 is a method of treating a disease in a subject in need thereof, comprising:

administering to the subject a therapeutically effective amount of each of m pharmaceutical compositions, each comprising at least one of n bacterial strains, wherein the n bacterial strains comprise at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain;

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n bacterial strains comprises a toxin system;

Embodiment 146 is a method of treating a disease in a subject in need thereof, comprising:

wherein each of the second through nth bacterial strains has a previous bacterial strain;

wherein n is at least 3;

wherein each of the n strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein the lytic plasmid of each of the n bacterial strains comprises a toxin system;

Wherein each of the second through nth bacterial strains independently produces a first toxin/first antitoxin pair and a second antitoxin from a second toxin/second antitoxin pair produced by a previous bacterial strain, wherein the first toxin is effective against the previous bacterial strain;

Embodiment 147 is the method of embodiment 145 or 146, wherein n is 3.

Embodiment 148 is the method of embodiment 145 or 146, wherein n is 4, 5, 6, 7, 8, 9, or 10.

Embodiment 149 is the method of any one of embodiments 145-146, wherein m ═ n.

Embodiment 150 is the method of any one of embodiments 141-149, wherein each of the toxin systems is independently encoded in the genome, on a plasmid, on multiple plasmids, or a combination thereof.

Embodiment 151 is the method of any one of embodiments 141-150, wherein each of the second through nth bacterial strains does not produce the second toxin of the toxin system of the previous strain and the first bacterial strain does not produce the fourth toxin of the toxin system of the nth bacterial strain.

Embodiment 152 is the method of any one of embodiments 145-151, wherein administering comprises sequentially administering to the subject each of the n bacterial strains or the m pharmaceutical compositions.

Embodiment 153 is the method of embodiment 152, wherein administering comprises administering each of the m pharmaceutical compositions simultaneously, and wherein each of the m pharmaceutical compositions has a different release profile that releases the first, second, and third bacterial strains.

Embodiment 154 is the method of any one of embodiments 141-153, wherein each of the first, second, and third bacterial strains expresses a different therapeutic agent.

Embodiment 155 is the method of any one of embodiments 141-154, wherein the disease is cancer or an infection.

Embodiment 156 is the method of embodiment 155, wherein the infection is caused by an infectious agent selected from the group consisting of: campylobacter jejuni (Campylobacter jejuni), Clostridium botulinum (Clostridium botulinium), Escherichia coli (Escherichia coli), Listeria monocytogenes (Listeria monocytogenes), and Salmonella (Salmonella).

Embodiment 157 is the method of embodiment 155, wherein the cancer is selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular carcinoma, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer.

Embodiment 158 is the method of any one of embodiments 1-83 or 134-157, the bacterial strain of any one of embodiments 84-111, the pharmaceutical composition of any one of embodiments 112-113, the system of any one of embodiments 114-123, 132, or 133, or the kit of any one of embodiments 124-131, wherein the one or more bacterial strains express the therapeutic agent.

Embodiment 159 is the method of any one of embodiments 1-83 or 134-157, the bacterial strain of any one of embodiments 84-111, the pharmaceutical composition of any one of embodiments 112-113, the system of any one of embodiments 114-123, 132, or 133, or the kit of any one of embodiments 124-131, wherein the one or more bacterial strains express a therapeutic agent selected from the group consisting of: inhibitory nucleic acids, cytokines, enzymes, peptide hormones, fusion proteins, clotting factors, toxins, antimicrobial peptides, and antibodies or antigen-binding fragments thereof.

Embodiment 160 is a method, comprising:

culturing a first bacterial strain in a growth environment for a first period of time;

adding a second bacterial strain to the growth environment and culturing the second bacterial strain for a second period of time;

Adding a third bacterial strain to the growth environment and culturing the third bacterial strain for a second period of time;

wherein each of the first, second and third strains comprises a toxin system;

wherein the toxin system is encoded on a plasmid, a plurality of plasmids, or integrated into the host genome, or a combination thereof.

Embodiment 161 is the method of embodiment 160, wherein each of the first, second, and third bacterial strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

An activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein the quorum sensing molecule may be the same or different in each of the first, second and third bacterial strains;

wherein the quorum sensing molecule of each of the first, second and third strains may or may not each have an effect on the activatable promoter of the lytic gene of the other strain.

Embodiment 162 is the method of embodiment 161, wherein each lytic plasmid of the first, second, and third bacterial strains is a plasmid comprising a toxin system of the bacterial strain, or wherein each activator plasmid of the first, second, and third bacterial strains is a plasmid comprising a toxin system of the bacterial strain, or wherein the toxin system of each bacterial strain is integrated into the genome.

Embodiment 163 is a method of culturing n bacterial strains, comprising:

culturing a first bacterial strain of the n bacterial strains in a culture for a first period of time;

Adding a second bacterial strain of the n bacterial strains to the growth environment and culturing the second bacterial strain for a second period of time;

adding each of the other n bacterial strains to a growth environment and culturing each of the bacterial strains over a period of time;

wherein n is an integer of three or more, and

wherein the plasmid of each of the n bacterial strains comprises a toxin system;

wherein the toxin system of the first bacterial strain produces a first toxin/first antitoxin pair and an nth antitoxin from the nth toxin/nth antitoxin pair, wherein the first bacterial strain does not produce the nth toxin;

wherein the toxin system of each mth plasmid produces an mth toxin/mth antitoxin pair, and an (m-1) th antitoxin from the (m-1) th toxin/(m-1) th antitoxin pair, wherein m is an element of the set {2, 3, …, n } of the other strains of the n bacterial strains.

Embodiment 164 is the method of embodiment 163, wherein each of the n bacterial strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

Wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein the quorum sensing molecule may be the same or different in each of the n bacterial strains;

wherein each quorum-sensing molecule of each of the n strains may or may not have an effect on an activatable promoter of a lytic gene of another of the n strains.

Embodiment 165 is the method of embodiment 164, wherein each lytic plasmid of the n strains is a plasmid comprising a toxin system of the strain.

Embodiment 166 is the method of any one of embodiments 163-164, wherein each of the n bacterial strains produces a payload.

Embodiment 167 is the method of any one of embodiments 163-165, wherein each of the n bacterial strains produces a different payload.

Embodiment 168 is the method of any one of embodiments 163-165, wherein each of the n bacterial strains produces the same payload.

Embodiment 169 is the method of any one of embodiments 166-168, wherein each payload is therapeutic.

Embodiment 170 is the method of embodiment 167, wherein the payload of the mth bacterial strain produces the mth substrate by acting directly or indirectly on the substrate of the (m-1) th bacterial strain, and wherein the payload of the first strain acts on the substrate present in the environment in which the n bacterial strains are cultured.

Embodiment 171 is the method of any one of embodiments 163-170, wherein n is 4.

Embodiment 172 is the method of any one of embodiments 163-170, wherein n is 5.

Embodiment 173 is the method of any one of embodiments 163-170, wherein n is 6.

Embodiment 174 is the method of any one of embodiments 160-162, further comprising culturing or co-culturing one or more additional bacterial strains in the culture.

Embodiment 175 is the method of embodiment 174, wherein each of the one or more additional bacterial strains comprises:

a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

the quorum sensing molecule is different in each of the first, second, and third bacterial strains and the one or more additional bacterial strains;

wherein the quorum sensing molecule of each of the first, second and third strains and the one or more additional bacterial strains each has no or substantially no effect on an activatable promoter of a lytic gene of another bacterial strain used in the culture.

Embodiment 176 is the method of embodiment 175, wherein the lytic plasmid of each of the one or more additional bacterial strains comprises a toxin system.

Embodiment 177 is the method of any one of embodiments 160-162 or 174-176, wherein the lytic plasmid and the activator plasmid of each of the at least first, second and third bacterial strains are the same plasmid.

Embodiment 178 is the method of any one of embodiments 160-162 or 174-176, wherein the lytic plasmid and the activator plasmid of each of the at least first, second and third bacterial strains are independent plasmids.

Embodiment 179 is the method of any one of embodiments 160-162 or 174-178, wherein at least the first, second, and third bacterial strains are in metabolic competition.

Embodiment 180 is the method of any one of embodiments 160-162 or 174-179, wherein at least the first, second and third bacterial strains are selected from escherichia coli, salmonella typhimurium or bacterial variants thereof.

Embodiment 181 is the method of any one of embodiments 160-162 or 174-180, wherein at least each of the first, second and third bacterial strains has no growth advantage over the other strain in the culture.

Embodiment 182 is the method of any one of embodiments 160-162 or 174-181, wherein in each of the at least first, second and third bacterial strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene.

Embodiment 183 is the method of any one of embodiments 160-162 or 174-182, wherein the lytic gene in at least each of the first, second and third bacterial strains is E from bacteriophage Φ X174.

Embodiment 184 is the method of any one of embodiments 160-162 or 174-183, wherein the activatable promoter in at least each of the first, second and third bacterial strains is a LuxR-AHL activatable luxI promoter and the activator gene is LuxI.

Embodiment 185 is the method of embodiment 182, wherein the at least one reporter gene is selected from a gene encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), or variants thereof.

Embodiment 186 is the method of embodiment 182, wherein the degradation label is an ssrA-LAA degradation label.

Embodiment 187 is the method of any one of embodiments 160-162 or 174-186, wherein at least one of the plasmids is integrated into the genome of at least one of the first, second and third bacterial strains.

Embodiment 188 is the method of any one of embodiments 160-162 or 174-187, wherein the culturing is performed in a specified growth environment.

Embodiment 189 is the method of any one of embodiments 160-162 or 174-188, wherein the culturing is performed in a microfluidic device.

Embodiment 190 is the method of any one of embodiments 160-162 or 174-188, wherein the culturing is performed in a bioreactor.

Embodiment 191 is the method of any one of embodiments 160-162 or 174-188, wherein the culturing is performed in vivo.

Embodiment 192 is the method of any one of embodiments 160-191, wherein each bacterial strain produces the therapeutic payload and is cultured in a human or animal patient in need of the therapeutic payload.

Embodiment 193 is the method of any one of embodiments 160-162 or 174-191, wherein each of the first, second, and third time periods ranges from about 12 hours to about 72 hours.

Embodiment 194 is the method of any one of embodiments 160, 174, 191, or 193, wherein each of the first, second, and third time periods ranges from about 1 hour to about n hours.

Embodiment 195 is the method of any one of embodiments 160-162, 174-191, or 193, wherein the first, second, and third time periods are each selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours.

Embodiment 196 is the method of any one of embodiments 163-173, wherein each of the n time periods partially or completely overlap.

Embodiment 197 is the method of any one of embodiments 163-173 or 196, wherein the n bacterial strains are cultured together.

Embodiment 198 is the method of any one of embodiments 163-173, 196 or 197, wherein each of the n bacterial strains is added to the culture sequentially such that each mth bacterial strain is added to the culture after passage for the (m-1) th time period.

Embodiment 199 is the method of any one of embodiments 160-.

Embodiment 200 is a method of maintaining a co-culture, the method comprising:

co-culturing at least three bacterial strains;

wherein each of the at least three bacterial strains comprises a lytic plasmid having a lytic gene under the control of an activatable promoter; and

an activator plasmid having an activator gene, expression of which promotes accumulation of quorum sensing molecules;

wherein the activatable promoter of the lytic gene and the expression of the activator gene are both activated by the quorum sensing molecule;

wherein the quorum sensing molecule is different in each of the at least three bacterial strains;

wherein the quorum sensing molecule of each of the at least three strains has no or substantially no effect on the activatable promoter of the lytic gene of another strain;

wherein the lytic plasmid comprises a toxin/antitoxin system.

Embodiment 201 is the method of embodiment 200, wherein the toxin/antitoxin system produces a toxin/antitoxin pair and a different antitoxin of another strain.

Embodiment 202 is the method of embodiment 200 or 201, wherein the lytic plasmid and the activator plasmid of each of the at least three strains are the same plasmid.

Embodiment 203 is the method of any one of embodiments 200-202, wherein the lytic plasmid and the activator plasmid of each of the at least three bacterial strains are independent plasmids.

Embodiment 204 is the method of any one of embodiments 200-203, wherein at least three of the bacterial strains are metabolically competitive.

Embodiment 205 is the method of any one of embodiments 200-204, wherein the at least three bacterial strains are selected from escherichia coli, salmonella typhimurium, or bacterial variants thereof.

Embodiment 206 is the method of any one of embodiments 200-205, wherein each of the at least three bacterial strains has no growth advantage over the other strain.

Embodiment 207 is the method of any one of embodiments 200-206, wherein, in each of the at least three bacterial strains, the lytic plasmid comprises a lytic gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, an optional degradation tag, and an optional reporter gene.

Embodiment 208 is the method of any one of embodiments 200-207, wherein the lytic gene in the at least three bacterial strains is E from bacteriophage Φ X174.

Embodiment 209 is the bacterial strain of any one of embodiments 200-208, wherein the activatable promoter is a LuxR-AHL activatable luxI promoter and the activator gene is LuxI.

Embodiment 210 is the method of embodiment 207, wherein the at least one reporter gene is selected from a gene encoding Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), or variants thereof.

Embodiment 211 is the method of embodiment 207, wherein the degradation tag is an ssrA-LAA degradation tag.

Embodiment 212 is the method of any one of embodiments 200-211, wherein the co-culture is inoculated at a ratio of 1:1:1 for each of the at least three bacterial strains.

Embodiment 213 is the method of any one of embodiments 200-212, wherein at least one of the plasmids is integrated into the genome of at least one of the at least three bacterial strains.

Embodiment 214 is the method of any one of embodiments 200-213, wherein the culturing is performed in a microfluidic device.

Embodiment 215 is the method of any one of embodiments 200-214, wherein the period of time is 12 to 72 hours.

Embodiment 216 is the method of any one of embodiments 200-214, wherein the period of time is selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours.

Embodiment 217 is the method of any one of embodiments 200-214, wherein the period of time is selected from the group consisting of 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours.

Embodiment 218 is the method of any one of embodiments 200-217, wherein the co-culture of the at least three strains is in a constant lytic state; wherein the constant lytic state is characterized by a steady state balance of growth and lysis of the at least three bacterial strains.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

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