阅读说明：本技术 一种基于铜绿假单胞菌群体感应系统的基因开关系统及其应用 (It is a kind of based on the gene switching system of pseudomonas aeruginosa intervention school-based and its application ) 是由 吴俊俊 周朋 包美娇 董明盛 于 2019-06-28 设计创作，主要内容包括：本发明公开了一种基于铜绿假单胞菌群体感应系统的基因开关系统及其应用,该基因开关系统包括铜绿假单胞菌的信号分子蛋白基因lasI,受体蛋白基因lasR和可以感应信号分子受体蛋白复合体的启动子序列PlasI。本发明基因开关系统是一种不依赖诱导剂,可以自发动态调节靶基因表达的基因开关系统,并利用该系统构建可以在合适的OD<Sub>600</Sub>状态下启动靶基因表达的发酵工程菌。本发明将基因元件分别构建到不同的表达载体上,本发明在工程菌内部构建了一个基因开关,具有自发动态调节发酵菌株代谢流,控制基因表达的功能,可以避免诱导剂的添加,节约发酵成本,简化发酵流程。(The invention discloses a kind of based on the gene switching system of pseudomonas aeruginosa intervention school-based and its application, the gene switching system include pseudomonas aeruginosa signaling molecule protein gene lasI, receptor protein gene lasR and can be with the promoter sequence PlasI of induction signal molecule receptor protein complex.Gene switching system of the present invention is that one kind does not depend on inducer, can be with the gene switching system of spontaneous dynamic regulation expression of target gene, and being constructed using the system can be in suitable OD 600 Start the fermentation of expression of target gene under state.Genetic elements are building up on different expression vectors by the present invention respectively, and the present invention has spontaneous dynamic regulation fermentation strain metabolic fluxes in one gene switching of engineering bacteria internal build, control the function of gene expression, fermentation costs can be saved to avoid the addition of inducer, simplify fermentation process.)

1. one kind is based on the gene switching system of pseudomonas aeruginosa (Pseudomonas aeruginosa) intervention school-based, It is characterized in that, the system element includes signaling molecule the protein gene lasI, receptor protein gene lasR of pseudomonas aeruginosa With can be with the promoter sequence PlasI of induction signal molecule receptor protein complex.

2. according to claim 1 be based on pseudomonas aeruginosa (Pseudomonas aeruginosa) intervention school-based Gene switching system, which is characterized in that the signaling molecule protein gene lasI and receptor protein gene lasR composing type turn Record translation synthesizes corresponding signaling molecule albumen and receptor protein, signaling molecule albumen can be with composite signal molecules, when in environment Signaling molecule run up to after a certain concentration and receptor protein combines and forms signaling molecule-receptor protein complex, signaling molecule- Receptor protein complex can be integrated to PlasI promoter region and start the expression of downstream gene.

3. according to claim 1 be based on pseudomonas aeruginosa (Pseudomonas aeruginosa) intervention school-based Gene switching system, which is characterized in that described gene lasI, lasR, PlasI can be with transcription and translation composite signal molecule proteins With receptor protein and adjust expression of target gene.

4. a kind of expression vector, which is characterized in that be based on pseudomonas aeruginosa (Pseudomonas comprising described in claim 1 Aeruginosa) the gene switching system of intervention school-based.

5. expression vector according to claim 1, which is characterized in that the expression vector will be based on pseudomonas aeruginosa The gene switching system of (Pseudomonas aeruginosa) intervention school-based, which is preferably building up on expression vector, to be convenient for Duplication and expression in microbial body.

6. a kind of genetic engineering bacterium, which is characterized in that be based on pseudomonas aeruginosa comprising described in claim 1 The gene switching system of (Pseudomonas aeruginosa) intervention school-based or expression as claimed in claim 4 carry Body.

7. genetic engineering bacterium according to claim 1, which is characterized in that the genetic engineering bacterium utilizes false single based on verdigris The gene switching system of born of the same parents bacterium (Pseudomonas aeruginosa) intervention school-based realizes egfp expression Dynamic regulation.

8. a kind of described in claim 1 based on pseudomonas aeruginosa (Pseudomonas aeruginosa) intervention school-based Gene switching system in real time adjust fermentation strain metabolic fluxes, control the application in the function of gene expression.

9. application according to claim 8, which is characterized in that the gene switching system is in the work switched with quiding gene Journey bacterium is that fermentation strain ferments, and the incipient stage does not start the expression of target gene, as fermentation liquid OD₆₀₀Start target when reaching 0.5 The transcription and translation of gene.

Technical field

The invention belongs to genetic engineering fields, and in particular to one kind is based on pseudomonas aeruginosa (Pseudomonas Aeruginosa the gene switching system of intervention school-based) and its application.

Background technique

Fermentation needs to synthesize a large amount of recombinant proteins, builds synthesis access, if begun to largely in culture starting point Resource intracellular can be competed with cell metabolism by expressing recombinant protein, bring huge burden to thallus, metabolism network is unbalance to be will lead to The accumulation of some toxic intermediate products also can cause to poison, bacterial strain is made to cannot keep good growth conditions to thalli growth, Influence the yield of final product.Therefore forefathers explore a variety of operating elements, realize the induction regulating controlling to gene, comprising: color ammonia Sour operon, arabinose operon and many heterozygosis operons etc., wherein with the lactose operon application induced by IPTG The most extensively.Inducible promoter has many advantages, including can adjust Gene expression intensities by adjusting inducer concentrations, pass through The starting time of inducer addition time control gene expression is controlled to balance the growth of thallus and the synthesis of product etc..But it lures There is also many problems for conductivity type promoter, including cannot achieve the dynamic regulation of target gene, can not timely feedbacking to cell state Deng.In addition, the price of inducer valuableness hinders its application in large-scale industrial production.

One of solution to this problem is exactly to introduce dynamic gene switching in fermentation strain to carry out controlling soil moist base The expression of cause, there are many trials of this respect at present.But these strategies are needed in response to specific intermediate product, cell State or culture medium composition, lack universality, can not cope with product requirements complicated and changeable.And quorum sensing (Quorum Sensing) system has the advantages that dynamic regulation and versatility as the monitoring induction system of cell density simultaneously.Therefore, base There is very high researching value and application prospect in the dynamic adjusting system of quorum sensing.

Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a kind of common pathogen, belongs to gram-negative Property, its vital movement process is regulated and controled by the intervention school-based that homoserine lactone mediates.Expression, life including virulence factor The formation of object film, the expression of antibiotic discharge pump and motility etc..The intervention school-based of pseudomonas aeruginosa is Fei Shi arc The extension of bacteria quorum sensing system research, pseudomonas aeruginosa intervention school-based related elements and Fermi operator LuxI/R system There is significant homology, but the intervention school-based of pseudomonas aeruginosa is increasingly complex.P. aeruginosa bacteria quorum sensing is One complicated hierarchical network contains two sets of intervention school-baseds of Las and Rhl, they are mutually indepedent and interrelated.Las In system, lasi gene controls signaling molecule 3OC₁₂The formation of-HSL (PAI 1) is secreted into extracellular ring after signaling molecule synthesis Border, after accumulation is to certain concentration can and LasR combine and activate a large amount of virulence factor genes expression.Include: lasB, lasA, Apr, toxA and lasI itself, experiments have shown that the pseudomonas aeruginosa for lacking active LasR albumen is harmless to animal.It grinds Study carefully by real-time fluorescence quantitative PCR explore pseudomonas aeruginosa controlled by intervention school-based it is related to biomembrane synthesis Gene, the synthesis of discovery algD, pslA, pelA, LasI, lasR, RhlA etc. series of genes and biomembrane is closely related.

In Rhl system, rhl is catalyzed the synthesis of N-butyryl-L-HSL (PAI2), and the signaling molecule and RhlR can after combining To activate the rhlAB gene for being responsible for rhamnolipid synthesis, the rhli gene for being responsible for the synthesis of signaling molecule albumen and lasB gene Expression.Rhl system is also responsible for some virulence factor expression, including pyo, cyanide and chitin etc..The two systems it Between there are Cascade Regulation, Las system controls the expression of transcription activating protein RhlR, therefore the gene of Rhl system control needs Complete and active Las system can be just fully active.Two kinds of systems adjust several genes expression, including synthesis elasticity simultaneously Protease, secretory protein, catalase, exotoxin, outer agglutinin, acyl homoserine lactones and superoxide dismutase etc. The expression of a variety of virulence factor genes.PAI I can hinder the combination of PAI II and RhlR, it is ensured that two systems are respectively suitable Time operation.

The now pseudomonas aeruginosa lasI/lasR intervention school-based about report in the art, is generally used to do Evil bacterial examination is surveyed, food antiseptic or biomembrane and food spoilage etc., Shobharani etc. are single for the vacation for causing milk corrupt Born of the same parents bacterium utilizes the signal communication of pseudomonad in 2 (H) furanone blocking fermentation milk of 300 μm of ol/L concentration, it is suppressed that its poison Shelf life extension has been arrived 9d by the generation of the power factor.Jamuna etc. have studied cumin, turmeric, absinth, rhizoma corydalis, fragrant nutmeg, Quorum sensing that the fragrance such as faenum graecum and cardamom essential oil mediates acylated homoserine lactone and biofilm formation It influences, there is discovery cumin essential oil when concentration is 0.02% volume ratio best QS inhibitory effect and antibiont film to form work Property, the cumin essential oil of subinhibitory concentration inhibits vacation by block cell contact, reduction cell metabolism and extracellular polymeric generation The biofilm formation of monad delays corruption caused by crymophilia pseudomonad PSPF19 in freezing milk.But utilize copper Green pseudomonad intervention school-based can detecte the characteristic of cell concentration application there is not been reported.

Summary of the invention

Goal of the invention: in view of the problems of the existing technology, the present invention provides a kind of based on pseudomonas aeruginosa The gene switching system of the intervention school-based of (Pseudomonas aeruginosa), the gene switching system are that one kind is disobeyed Rely inducer, can be with the gene switching system of spontaneous dynamic regulation expression of target gene, and being constructed using the system can be suitable OD₆₀₀Start the genetic engineering bacterium of expression of target gene under state.

Genetic elements by being building up on different expression vectors by the present invention respectively, and the present invention is in engineering bacteria internal build One gene switching has spontaneous dynamic regulation fermentation strain metabolic fluxes, controls the function of gene expression, can be to avoid induction Fermentation costs are saved in the addition of agent, simplify fermentation process.

Technical solution: to achieve the goals above, a kind of as described herein to be based on pseudomonas aeruginosa The gene switching system of (Pseudomonas aeruginosa) intervention school-based, which is characterized in that the system element includes Signaling molecule the protein gene lasI, receptor protein gene lasR of pseudomonas aeruginosa and can be with induction signal molecule receptor protein The promoter sequence PlasI of complex；Its nucleotide sequence such as SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 institute Show.

Wherein, signaling molecule protein gene lasI and receptor protein gene lasR composing type transcription and translation synthesize corresponding letter Number molecule protein and receptor protein, signaling molecule albumen can be with composite signal molecule, when the signaling molecule in environment runs up to one Determine after concentration and receptor protein combines and forms signaling molecule-receptor protein complex, signaling molecule-receptor protein complex can be tied It closes PlasI promoter region and starts the expression of downstream gene.

Preferably, described gene lasI, lasR, PlasI can be with transcription and translation composite signal molecule proteins and receptor egg It is white and adjust expression of target gene.

Expression vector of the present invention includes described based on pseudomonas aeruginosa (Pseudomonas Aeruginosa) the gene switching system of intervention school-based.The present invention is based on pseudomonas aeruginosa intervention school-based genes The expression vector of switching system includes support C-PlasI-egfp, the A-trc (lost formed by PlasI, lasI, lasR Operon)-lasR and E-trc (lost operon)-lasI.

Wherein, the expression vector will be based on pseudomonas aeruginosa (Pseudomonas aeruginosa) quorum sensing The gene switching system of system is building up on expression vector to replicate and express in microbial body

Genetic engineering bacterium of the present invention, comprising described based on pseudomonas aeruginosa (Pseudomonas Aeruginosa) the gene switching system of intervention school-based or the expression vector.The present invention is based on P. aeruginosas The genetic engineering bacterium of bacteria quorum sensing system gene switching system is mainly by expression vector C-PlasI-egfp, A-trc (lost Operon)-lasR and E-trc (lost operon)-lasI is together in quiding gene engineering bacteria.

Wherein, the genetic engineering bacterium can use described based on pseudomonas aeruginosa (Pseudomonas Aeruginosa) the gene switching system of intervention school-based, realizes the dynamic regulation of egfp expression.

Base of the present invention based on pseudomonas aeruginosa (Pseudomonas aeruginosa) intervention school-based Because switching system is adjusting fermentation strain metabolic fluxes in real time, the application in the function of gene expression is controlled.

Wherein, the gene switching system does not start the expression of target gene in fermentation starting point, as fermentation liquid OD₆₀₀Reach 0.5 The transcription and translation of Shi Qidong target gene.

To import containing based on pseudomonas aeruginosa (Pseudomonas aeruginosa) quorum sensing system in the present invention The engineering bacteria of the gene switching system of system is the fermentation that fermentation strain carries out.

The present invention overcomes the dependence in existing fermentation process to inducer, provides a kind of based on the sense of pseudomonas aeruginosa group Answer gene switching and its application of system.The present invention provides the Genetic elements sequence of gene switching, each constructing method of component with Be transferred to engineering bacteria method, based on pseudomonas aeruginosa gene switch work path and concrete application.

1, the present invention is the gene switching system based on pseudomonas aeruginosa intervention school-based, and the element of the system includes Signaling molecule protein gene lasI, receptor protein gene lasR and can be with signaling molecule-receptor protein complex specificity knot The promoter sequence PlasI of conjunction.Its nucleotide sequence is as shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5. LasI amino acid sequence is as shown in SEQ ID NO:2, and LasR amino acid is as shown in SEQ ID NO:4.

2, the present invention is based on the working principles of the gene switching of pseudomonas aeruginosa intervention school-based building: verdigris is false single Born of the same parents' bacterium signaling molecule protein gene and receptor protein gene are regulated and controled by constitutive promoter trc (lost operon), composing type table Up to signaling molecule albumen and receptor protein, signaling molecule proteins carry pseudomonas aeruginosa signaling molecule 3OC₁₂The synthesis of-HSL, When in environment signaling molecule and receptor protein concentration reach the detection threshold value of PlasI after, signaling molecule and receptor protein are answered Zoarium can be integrated to the region PlasI and start the expression of promoter downstream gene.

3, the present invention is based on the gene switching system of pseudomonas aeruginosa intervention school-based, start the mode of induction regulating controlling For the concentration of signaling molecule in induction environment, work as OD₆₀₀The enough promotor genes of the concentration of corresponding signaling molecule are opened when reaching 0.5 Close the expression of downstream target gene.

4, the gene switching systematic difference of the invention based on the building of pseudomonas aeruginosa intervention school-based has real When adjust fermentation strain metabolic fluxes, control the function of gene expression, can to avoid the addition of inducer, save fermentation costs, letter Change fermentation process.

The present invention can detecte the Characteristics Control foreign gene of cell concentration using pseudomonas aeruginosa intervention school-based Expression, whether gene, which expresses, is exactly influenced by bacterium is dense.The present invention is false single especially by verdigris is reconstructed in Escherichia coli body The intervention school-based of born of the same parents bacterium constructs the gene switching of adjustable expression of target gene, realizes the dynamic expressed recombinant protein Regulation.

The present invention constructs adjustable target by the intervention school-based of the reconstruct pseudomonas aeruginosa in Escherichia coli body The gene switching of gene expression, in fermentation starting point, RNA polymerase can not be integrated to promoter region, gene switching downstream it is green Color fluorescin is not expressed, genetic engineering bacterium persistent accumulation signaling molecule intracellular and receptor protein, when signaling molecule and receptor egg Signaling molecule-receptor protein complex is integrated to promoter region and RNA polymerase is induced to combine after white concentration reaches threshold value, opens The expression of dynamic green fluorescent protein, realizes the dynamic regulation to foreign protein.

The utility model has the advantages that compared with prior art, the present invention has the advantage that

The present invention constructs the gene switching system based on pseudomonas aeruginosa intervention school-based for the first time, and switch tool is real When adjust fermentation strain metabolic fluxes, control the function of gene expression.Present invention process science, method are simple, operation is easy；It can be with The addition of inducer is avoided, fermentation costs are saved, simplifies fermentation process.

Gene switching system of the invention is that one kind does not depend on inducer, can be with the base of spontaneous dynamic regulation expression of target gene Because of switching system, and can be in suitable OD using system building₆₀₀Start the genetic engineering bacterium of expression of target gene under state.

Genetic elements are building up on different expression vectors by the present invention respectively, and the present invention is in engineering bacteria internal build one A gene switching has spontaneous dynamic regulation fermentation strain metabolic fluxes, controls the function of gene expression, can be to avoid inducer Fermentation costs are saved in addition, simplify fermentation process.

Detailed description of the invention

Fig. 1 is that the gene switching based on pseudomonas aeruginosa intervention school-based constructs schematic diagram；

Fig. 2 is that the fluorescence intensity of gene switching starting egfp expression changes over time trend；

Fig. 3 is the fluorescence picture BL21 (C-PlasI-egfp) of confocal fluorescent microscopic shooting；

Fig. 4 is the fluorescence picture BL21 (E-trc (lost operon)-lasI) of confocal fluorescent microscopic shooting；

Fig. 5 is the fluorescence picture BL21 (A-trc (lost operon)-lasR) of confocal fluorescent microscopic shooting；

Fig. 6 is fluorescence picture BL21 (C-PlasI-egfp, the E-trc (lost of confocal fluorescent microscopic shooting Operon)-lasI, A-trc (lost operon)-lasR).

Specific embodiment

It is described further below in conjunction with drawings and examples.