Method for identifying protein of interest

文档序号：538563 发布日期：2021-06-01 浏览：5次中文

阅读说明：本技术 用于鉴定目的蛋白质的方法 (Method for identifying protein of interest ) 是由 M·布利兹纽克 M·里兹卡于 2019-10-17 设计创作，主要内容包括：本技术的公开内容涉及分析细胞或基因文库的方法,所述方法适用于所述文库的高通量分析。特别地,本技术涉及使用自动化机器人技术、信息技术、计算机和声学分配设备的高通量筛选的自动化方法,以鉴定目的蛋白质。(The disclosure of the present technology relates to methods of analyzing a cell or gene library that are suitable for high throughput analysis of the library. In particular, the present technology relates to automated methods of high throughput screening using automated robotics, information technology, computers, and acoustic distribution equipment to identify proteins of interest.)

1. A method of identifying a protein of interest, comprising:

(a) screening a first library of cells expressing proteins;

(b) identifying and selecting said cells expressing said protein having the desired function;

(d) arranging the solution of the second library into one or more microtiter plates;

(e) transferring a specified volume of each solution of the second library from the one or more microtiter plates onto one or more agar trays with an acoustic dispenser;

(f) incubating the one or more agar trays under conditions suitable for growing colonies on the one or more agar trays;

(g) picking the one or more colonies using a robot;

(h) transferring the one or more colonies to a liquid medium;

(i) growing the culture in the liquid medium and isolating DNA encoding the protein of interest having the desired function; and

(j) sequencing said DNA of said protein of interest, thereby identifying said protein of interest.

2. The method of claim 1, wherein the protein of interest is an enzyme, peptide, antibody or antigen-binding fragment thereof, protein antibiotic, fusion protein, vaccine or vaccine-like protein or particle, growth factor, hormone, or cytokine.

3. The method of claim 2, wherein the enzyme is selected from the group consisting of: amylase, xylanase, protease, glucoamylase, glucanase, mannanase, phytase, and cellulase.

4. The method of any one of the preceding claims, wherein the cell is a prokaryotic cell or a eukaryotic cell.

5. The method of claim 4, wherein the cell is a prokaryotic cell selected from a bacterial cell or an archaeal cell.

6. The method of claim 4, wherein the cell is a eukaryotic cell selected from a fungal cell, a yeast cell, an animal cell, or a plant cell.

7. The method according to any one of claims 1-4, wherein the cell is an E.coli cell, a Bacillus cell, a Trichoderma cell, a Torulopsis crassa (Komagataella) cell, an Aspergillus cell, a Thermomyces cell, or a Saccharomyces cell.

8. The method of any one of the preceding claims, wherein the desired function of the protein of interest is selected from the group consisting of: enzyme activity, substrate specificity, expression titer, and any combination thereof.

9. The method of any one of the preceding claims, wherein the one or more microtiter plates comprise wells, wherein the number of wells is selected from the group consisting of: 6. 12, 24, 48, 96, 384 or 1563.

10. The method of any one of the preceding claims, wherein in step (d) a dilution of the solution is prepared prior to arraying the second library of cells into the one or more microtiter plates.

11. The method of claim 10, wherein the dilution liquid is a serial dilution liquid, and wherein the dilution liquid is aligned in step (d).

12. The method of claim 11, wherein the serial dilution is repeated 5, 4, 3, or 2 times.

13. The method of any one of the preceding claims, wherein the solution is a liquid comprising a glycerol stock solution.

14. The method of any one of the preceding claims, wherein the specified volume transferred from the one or more microtiter plates onto the one or more agar trays in step (e) using the acoustic dispenser is selected from 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8, nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, 30nL or more.

15. The method of any one of the preceding claims, wherein in step (e), the one or more agar trays are positioned above the one or more microtiter plates, and wherein the one or more agar trays are oriented such that their agar surfaces face the one or more microtiter plates.

16. The method of any one of the preceding claims, wherein the incubation time in step (f) is from 12 hours to 120 hours.

17. The method of any one of the preceding claims, wherein the incubation temperature in step (f) is from 15 ℃ to 50 ℃.

Technical Field

The present technology relates to methods suitable for high throughput screening or analysis of cell or gene libraries. In particular, the present technology relates to automated methods for high throughput screening of cell or gene libraries to identify library members using automated robotics, information technology, computers, and acoustic distribution equipment. Libraries and library members may contain microorganisms, unicellular organisms, prokaryotes, bacteria, and archaea; multicellular organisms, eukaryotes, fungi, yeasts, animal and plant cells. Libraries and library members may contain nucleic acids, nucleotides, polynucleotides, genes, RNA or DNA encoding proteins, polypeptides, and enzymes. The analysis of the library and library members may be the identification of the function of a protein, polypeptide or enzyme, or the identification of the sequence of a polynucleotide, gene, RNA, DNA, polypeptide, protein or enzyme.

Background

The following description is provided to assist the reader in understanding. None of the information provided or references cited is admitted to be prior art.

High Throughput Screening (HTS) refers to screening large numbers of samples for a desired molecule in a short time and is widely used in fields such as drug discovery, enzyme biology, and other biological, biochemical, and chemical fields. High throughput screening can rapidly handle millions of chemical, genetic or pharmacological tests by using robotics, data processing/control software, liquid handling equipment and sensitive detectors. HTS programs allow the rapid identification of active compounds, antibodies or genes that modulate a particular biomolecular pathway. The results of these experiments provide a starting point for drug design and understanding of the interaction or utility of specific biochemical processes in biology.

One example of High Throughput Screening (HTS) is enzyme evolution, also known as directed evolution, a method used in protein engineering that mimics the process of natural selection to direct the development of proteins or nucleic acids to user-defined targets. Such a target may be, for example, a certain function exhibited by the enzyme, an improved function, or other desired qualities. Directed evolution involves subjecting genes to repeated rounds of mutagenesis (creating a library of gene variants), selection (expressing these gene variants recombinantly in cells, thereby creating a library of cells expressing the gene variants, and isolating library members expressing gene variants with the desired function) and amplification (generating a nucleic acid template for the next round of analysis). Directed evolution can be used for protein engineering, as an alternative method for rationally designing and modifying proteins, and can also be used for researching the basic evolution principle in a controlled laboratory environment.

In addition to directed evolution of proteins or nucleic acids, high throughput screening can similarly be used to identify or analyze libraries of nucleic acids, such as DNA and RNA, peptides, polypeptides, and proteins (e.g., enzymes) to obtain desired attributes, all of which can be recombinantly expressed in cells to create libraries of cells. Cells commonly used in high throughput screening are prokaryotic, such as bacteria and archaea, and eukaryotic, such as fungi (e.g., yeast), and animal and plant cell lines.

Despite the improvements in high throughput screening techniques and the equipment required to perform these techniques for screening biological materials, cells, nucleic acids and proteins in recent years, these techniques are still complex, sometimes requiring manual intervention to perform certain activities. For example, steps that typically must be performed manually include cell plating, picking single cell colonies (hit picking), growing and propagating these picked cells, and isolating and sequencing the DNA or protein of interest. Such processes performed manually by humans during high throughput screening are costly, slow and prone to error. Therefore, there is a need in the art to reduce the cost, time required and frequency of errors in performing high throughput screening of biological materials by automation.

Disclosure of Invention

Disclosed herein are fully automated workflow methods for high throughput screening of libraries and identification of library members based on specific properties (e.g., expression of a desired protein function, such as enzyme activity, substrate specificity, enzyme stability, and/or expression titer).

In one aspect, the present disclosure provides a method for identifying a protein of interest, the method comprising: (a) screening a first library of cells expressing proteins; (b) identifying and selecting cells expressing a protein having a desired function; (c) generating a second library comprising cells expressing proteins with desired functions; (d) arranging the solution of the second library into one or more microtiter plates; (e) transferring a specified volume of each solution of the second library from one or more microtiter plates onto one or more agar trays with an acoustic dispenser; (f) incubating the one or more agar trays under conditions suitable for growing colonies on the one or more agar trays; (g) picking one or more colonies using a robot; (h) transferring one or more colonies to a liquid medium; (i) growing the culture in a liquid medium and isolating DNA encoding the protein of interest having the desired function; and (j) sequencing the DNA of the protein of interest, thereby identifying the protein of interest.

In some embodiments, the protein of interest is an enzyme, peptide, antibody or antigen-binding fragment thereof, protein antibiotic, fusion protein, vaccine or vaccine-like protein or particle, growth factor, hormone, or cytokine.

In some embodiments, the enzyme is selected from the group consisting of: amylase, xylanase, protease, glucoamylase, glucanase, mannanase, phytase, and cellulase.

In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell selected from a bacterial cell or an archaeal cell. In some embodiments, the cell is a eukaryotic cell selected from a fungal cell, a yeast cell, an animal cell, or a plant cell. In some embodiments, the cell is an escherichia coli cell, a bacillus cell, a trichoderma cell, a coltsia (Komagataella) cell, an aspergillus cell, a thermomyces cell, or a yeast cell.

In some embodiments, the desired function of the protein of interest is selected from the group consisting of: enzyme activity, substrate specificity, expression titer, and any combination thereof.

In some embodiments, one or more microtiter plates comprise wells, wherein the number of wells is selected from the group consisting of: 6. 12, 24, 48, 96, 384 or 1563.

In some embodiments, in step (d), a dilution of the solution is prepared prior to arraying the second library of cells into one or more microtiter plates. In some embodiments, the dilution is a serial dilution, and wherein the dilutions are arranged in step (d). In some embodiments, the serial dilutions are repeated 5, 4, 3, or 2 times.

In some embodiments, the solution is a liquid comprising a glycerol stock.

In some embodiments, the specified volume transferred from the one or more microtiter plates to the one or more agar trays in step (e) using an acoustic dispenser is selected from 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8, nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, 30nL or more.

In some embodiments, in step (e), the one or more agar trays are positioned above the one or more microtiter plates, wherein the one or more agar trays are oriented such that their agar surfaces face the one or more microtiter plates.

In some embodiments, the incubation time in step (f) is 12 hours to 120 hours. In some embodiments, the incubation temperature in step (f) is from 15 ℃ to 50 ℃.

Drawings

Fig. 1 is a schematic diagram depicting the basic operation of an exemplary acoustic dispenser (an Acoustic Transfer System (ATS) from EDC Biosystems). The source wells in the microtiter plate are centered over an acoustic horn that generates acoustic energy. This energy flows through the coupling fluid, the bottom of the microtiter plate, and the solution in the wells of the microtiter plate, creating waves in the wells, causing the solution present in the wells to form a single droplet. The receiving wells or surfaces (e.g., agar trays) are inverted over the source wells, and a single droplet travels vertically from the source well until it lands in the corresponding receiving well or surface.

FIG. 2 is an agar tray on which 32 E.coli library members are spotted. According to the protocol of example 2, each member was spotted at three incremental dilutions (left to right) to obtain a single colony.

Fig. 3 is a close-up of a member of the pichia pastoris (p. pastoris) library transferred to an agar tray at three incremental dilutions (from left to right) according to the protocol of example 2.

FIG. 4 is an agar tray with Pichia pastoris library members spotted according to example 3. Increasing dilution (top to bottom) is indicated. "1 x" corresponds to OD₆₀₀Is 1.

FIG. 5 shows sequencing data of amylase genes of interest from members of the Pichia pastoris library according to examples 1 and 4. Mixing the cultures resulted in multiple chromatographic peaks for residues 128, 256 and 433.

FIG. 6 shows sequencing data for individual colonies selected from the 1,000-fold dilution array of the mixed population in FIG. 4 described in examples 3 and 4, demonstrating that each of the three variants was recovered from the mixed culture after ATS transfer and growth, and that each of the three recovered variants was genetically pure.

Figure 7 provides images of two agar plates (library mix #1 and library mix # 2). These images show two libraries with different percentages of active variants.

Detailed Description

While the present technology will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.

I.Definition of

Before describing in detail exemplary embodiments of the present technology, definitions important for understanding the present technology are given. Unless otherwise indicated or apparent from the nature of the definitions, these definitions apply to all methods and uses described herein.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In the context of the present technology, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term generally means a deviation from the indicated value of ± 20%, preferably ± 15%, more preferably ± 10%, even more preferably ± 5%.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present technology, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If in the following a group is defined comprising at least a certain number of embodiments, this means also a group, which preferably consists of only these embodiments.

Furthermore, the terms "first," "second," "third," or "(a)", "(b)", "(c)", "(d)" and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the technology described herein are capable of operation in other sequences than described or illustrated herein, unless otherwise specifically indicated. Where the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii", etc. relate to steps of a method or use or assay, there is no continuity of time or time intervals between the steps unless otherwise indicated above or below the application, i.e. the steps may be performed simultaneously, or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between the steps.

It is to be understood that the present technology is not limited to the particular methodology, protocols, reagents, etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present technology which is limited only by the appended claims. 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.

As used herein, the term "library" means a collection of members, wherein at least two members of the library differ from each other in at least one characteristic, preferably in the nucleic acid sequence encoding a recombinantly expressed protein. In one embodiment, each member of the library differs from all other members of the library in at least one characteristic. Different members of the library may have the same nucleic acid sequence encoding the recombinantly expressed protein, as long as at least two members of the library differ from each other in the nucleic acid sequence encoding the recombinantly expressed protein. In particular embodiments, each member of the library differs from all other members of the library in the nucleic acid sequence encoding the recombinant protein. In one embodiment, each member of the library recombinantly expresses a variant of the parent protein. A variant may differ from a parent protein in one or more amino acid substitutions, insertions or deletions. In one embodiment, each member of the library recombinantly expresses a different variant of the parent protein. In one embodiment, at least two members of the library differ from each other in that they recombinantly express different variants of the same parent protein. For example, member 1 may express a variant of the parent protein a comprising the amino acid substitution S134A, and member 2 may express a variant of said parent protein a comprising the amino acid substitution G225C.

Libraries typically comprise 1,000 to 100,000 members, 5,000 to 80,000 members, 10,000 to 60,000 members, or 20,000 to 50,000 members.

Libraries can be provided by mutagenizing the amino acid sequence of a protein, also referred to as a parent protein, to produce different variants of the parent protein. Preferably, the amino acid sequence is randomly mutagenized. Techniques for random mutagenesis of proteins are well known to the skilled artisan and include Gene Site Saturation Mutagenesis (GSSM), described in Kretz et al (2004) Methods in enzymology (Methods Enzymol.) 388: 3-11. After mutagenesis, variants of a parent protein may be screened for a desired function, and only those variants having the desired function may be further analyzed by methods of the present technology. As discussed in further detail below, the desired functions include enzyme activity, substrate specificity, enzyme stability, and expression titer.

Within the present technology, a "member" of a library includes one or more cells that recombinantly express a protein. Preferably, one or more cells of a member are obtained from the same cell clone. Preferably, the members vary in only one aspect, which aspect is preferably related to the recombinantly expressed protein. That is, the members of the library are preferably all of the same cell type and vary only, for example, in the sequence of the recombinantly expressed protein or in the nucleic acid sequence encoding the recombinantly expressed protein.

The one or more cells comprised by a member or library may be prokaryotic or eukaryotic.

Exemplary prokaryotic cells are bacterial cells. Exemplary bacterial cells are E.coli cells and Bacillus cells, such as Bacillus subtilis cells or Bacillus licheniformis cells.

Exemplary eukaryotic cells are selected from the group consisting of fungal cells, animal cells, and plant cells. Exemplary fungal cells are yeast cells, trichoderma cells (e.g., trichoderma reesei), aspergillus cells (e.g., aspergillus niger cells), and thermomyces cells (e.g., thermophilus). Exemplary yeast cells are foal yeast cells (e.g., faffia foal (Komagataella phaffi)) (also known as Pichia pastoris) cells, and saccharomyces cells (e.g., yeast cells).

The term "protein" as used herein refers to a peptide, polypeptide, enzyme, antibody or antigen-binding fragment thereof, protein antibiotic, fusion protein, vaccine or vaccine-like protein or particle, growth factor, hormone or cytokine. All proteins have an amino acid sequence encoded by a nucleic acid sequence. Proteins are produced from genes by translational and transcriptional processes and may be further modified post-translationally. The protein may contain tags, leader peptides and/or other additional sequences. An "enzyme" is a protein that catalyzes a chemical reaction. Preferably, the enzyme is a hydrolase. Hydrolases are a class of enzymes that are commonly used as biochemical catalysts for breaking chemical bonds with water. Exemplary enzymes are phytase, protease, beta-glucanase, xylanase, mannanase, lipase, cellulase, glucoamylase, amylase, alpha-amylase and beta-amylase.

A "recombinantly expressed" protein refers to a protein produced by recombinant DNA techniques, e.g., a protein produced by a cell transformed with an exogenous DNA construct encoding a desired protein. Recombinantly expressed proteins from different members of the same library may be functionally or structurally related. Recombinantly expressed proteins from different members of the same library may be encoded by different alleles of the same gene. The recombinantly expressed proteins of different members of the same library may be homologues or orthologues of each other. Recombinantly expressed proteins of different members of the same library may be variants of a parent protein, e.g., variants generated by directed evolution. A variant may differ from a parent protein in one or more amino acids and may comprise a substitution, deletion or insertion of one or more amino acids as compared to the parent protein.

As used herein, the term "array" refers to a two-dimensional arrangement of features on a substrate surface. A "feature" can be a library, a library member, or a unit. The substrate on the surface of the agar is a tray. Arrays are typically composed of regular, ordered features, such as a rectilinear grid or parallel stripes; however, these features may also be disordered or random arrays.

As used herein, the term "array" means that the members of the library are arranged in a defined two-dimensional pattern and/or sequence in a microtiter plate or on the surface of an agar tray. Each defined well or spot of the array is associated with a particular member of the library. Preferably, each defined well or spot is also associated with a dilution value for its associated particular member, the dilution value for the undiluted member being zero. That is, each well or spot can be identified as a particular member at a particular dilution. The alignment may be performed manually or by automation (e.g., by a robot). In the context of the present technology, automated alignment is preferred. An exemplary robot for automated alignment includes, for example, a liquid handler, such as the 8-channel Vantage liquid handler of Hamilton (Hamilton).

The terms "microplate," "microtiter plate," "microwell plate," or "multiwell plate" are used interchangeably herein and refer to a plate having a plurality of "wells" for use as a small test vessel. Microtiter plates have become standard tools for analytical research and clinical diagnostic test laboratories. Microplates typically have 6, 12, 24, 48, 96, 384, 1536, 3456, or 9600 sample wells arranged in a 2:3 rectangular matrix. Each well of a microplate typically holds tens of nanoliters to several milliliters of liquid. The holes may be round or square in cross-section and have flat bottoms, round or pointed bottoms. Microtiter plates are made of glass or plastic and may be transparent or opaque. In one embodiment, the microtiter plate is a clear glass plate. The microtiter plate is compatible with acoustic dispensers. An exemplary microtiter plate comprises, for example, 384Greiner 781090.

An "agar tray" is a glass or plastic tray, the size of which corresponds to the size of a microtiter plate, and which is filled with a suitable culture medium solidified with agarose. The medium was solidified with an appropriate amount of agarose such that no medium dropped from the agar tray when the tray was inverted. The height of the agar in the agar tray is between 1 and 15mm, preferably between 2 and 13mm, more preferably between 3 and 10mm, and even more preferably between 4 and 8 mm. In one embodiment, the height of the agar in the agar tray is 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, or 15 mm. The height of the agar within the agar tray is selected such that the agar forms a substantially uniform surface and/or a surface that does not substantially dry during transfer of the cells to the agar tray. The height of the agar will affect the quality of the individual colonies formed in step (d) of the method of the present technology. An exemplary tray for filling with curing media includes, for example, Nunc OmniTrays.

"Medium" refers to a medium that includes any nutrients or other components necessary to support the growth of cells on or within the medium. The culture medium can be a liquid (referred to herein as "liquid medium"), semi-solid, or solid (e.g., a liquid medium solidified with agarose, such as 0.5%, 0.8%, 1.0%, 1.3%, 1.5%, 1.8%, or 2.0% agarose). The choice of medium will depend on the cell type or types present in the library and is known to those skilled in the art. The culture medium may contain a selection agent to select for those cells that contain a recombinant nucleic acid molecule that expresses a recombinantly expressed protein. An exemplary medium for pichia pastoris includes, for example, YPD, which contains 1% yeast extract, 2% peptone, and 2% glucose.

An "acoustic dispenser" refers to a machine in which localized acoustic energy is generated at a specific location corresponding to one or more well locations in a microtiter plate beneath the microtiter plate. The sonic energy transfers a designated volume of liquid from the pores created below. The volume is typically about 1 to 30 nanoliters, e.g., 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30 nL. The displaced liquid is pushed upwards by the sonic energy where it meets an agar tray, which is positioned above the microtiter plate with the agar side facing the open wells of the microtiter plate. The distance between the microtiter plate and the agar tray may be 1 to 20mm, for example 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm or 20 mm. The edges of the agar tray and the microtiter plate are aligned such that each spot on the agar tray can be directly associated with a well of the microtiter plate and, therefore, with a particular library member, preferably at a particular dilution. Fig. 1 shows an exemplary setup illustrating the basic operation of an acoustic splitter. Exemplary acoustic dispensers include, for example, the Echo liquid handler of ATS Gen5 or Labcyte of EDC biosystems.

The term "colony" refers to a visible cluster of cells growing on the surface or within a solid medium. In one embodiment, the colonies are derived from a single starting cell by cell division. "Single colony" refers to a colony that does not touch or overlap with adjacent colonies. For a single colony, the likelihood of the colony originating from a single cell rather than multiple cells is much higher. Thus, for subsequent analysis, e.g., by nucleic acid sequencing, a single colony is preferred because only one form of the molecule to be analyzed may be present in a single colony. To obtain individual colonies, dilution may be performed until there are not enough cells in a volume of liquid that, when transferred to an agar tray, the colonies generated by the individual cells are spaced far enough apart to avoid contact or overlap.

"incubation" refers to placing an inoculated solid or liquid medium, for example in an agar tray or microtiter plate, for a period of time and under conditions that promote cell growth and division. A specific set of conditions, e.g. specific temperature, humidity, CO₂Concentration, etc., will depend on the one or more cell types included in the library. In one embodiment, the incubation temperature is between 15 ℃ and 50 ℃, preferably between 20 ℃ and 45 ℃, more preferably between 25 ℃ and 40 ℃, and most preferably between 30 ℃ and 37 ℃. Similarly, the incubation period will depend on the growth and/or division rate of the cells under a given set of conditions. In one embodiment, the incubation time is from 12 to 120 hours, more preferably from 15 to 100 hours, even more preferably from 20 to 70 hours, and most preferably from 24 to 48 hours. In one embodiment, the incubation temperature is from 15 ℃ to 50 ℃ and the incubation time is from 12 to 120 hours. In another embodiment, the incubation temperature is from 20 ℃ to 45 ℃ and the incubation time is from 12 to 120 hours. In another embodiment, the incubation temperature is from 25 ℃ to 40 ℃ and the incubation time is from 12 to 120 hours. In another embodiment, the incubation temperature is 30 ℃ to 37 ℃ and the incubation time is 12 to 120 hours. In one embodiment, the incubation temperature is from 15 ℃ to 50 ℃ and the incubation time is from 15 to 100 hours. In another embodiment, the incubation temperature is from 20 ℃ to 45 ℃ and the incubation time is from 15 to 100 hours. In another embodiment, the incubation temperature is from 25 ℃ to 40 ℃ and the incubation time is from 15 to 100 hours. In another embodiment, the incubation temperature is from 30 ℃ to 37 ℃ and the incubation time is from 15 to 100 hours. In one embodiment, the incubation temperature is from 15 ℃ to 50 ℃ and the incubation time is from 20 to 70 hours.In another embodiment, the incubation temperature is from 20 ℃ to 45 ℃ and the incubation time is from 20 to 70 hours. In another embodiment, the incubation temperature is from 25 ℃ to 40 ℃ and the incubation time is from 20 to 70 hours. In another embodiment, the incubation temperature is from 30 ℃ to 37 ℃ and the incubation time is from 20 to 70 hours. In one embodiment, the incubation temperature is from 15 ℃ to 50 ℃ and the incubation time is from 24 to 48 hours. In another embodiment, the incubation temperature is from 20 ℃ to 45 ℃ and the incubation time is from 24 to 48 hours. In another embodiment, the incubation temperature is from 25 ℃ to 40 ℃ and the incubation time is from 24 to 48 hours. In another embodiment, the incubation temperature is 30 ℃ to 37 ℃ and the incubation time is 24 to 48 hours. Exemplary incubation times and temperatures include, for example, 30 ℃ overnight for E.coli and 30 ℃ for 48 hours for P.pastoris.

"picking a colony" refers to transferring some or all of the cells of the colony, preferably a single colony, to fresh solid or liquid media for propagation, or to a mixture of media and glycerol for storage. This is usually done by scraping with a ring or dipping with a stick or pipette tip. In the methods of the present technology, the colonies are picked using a robot that identifies the colonies, picks them and transfers them to new media. Robots for picking colonies are available from different companies and include RapidPick from Hadson Robotics, Inc. (Hudson Robotics)^TMAutomated colony picking System, Stannus machine from Singer Instruments, Pickolo from Scrobotics^TMColony selector and Molecular Devices Qpix 400 series microorganism colony selector

The phrase "nucleic acid" refers to an oligonucleotide, nucleotide, polynucleotide, or fragment of any of them, DNA or RNA of genomic, recombinant, or synthetic origin (e.g., mRNA, rRNA, tRNA), which may be single-stranded or double-stranded, and may represent a sense or antisense strand, a Peptide Nucleic Acid (PNA), or any DNA-like or RNA-like material of natural or synthetic origin, including, for example, iRNA (e.g., miRNA or siRNA), ribonucleoproteins (e.g., iRNP). The term includes nucleic acids, i.e., oligonucleotides, that contain known analogs of natural nucleotides. The term "nucleic acid sequence" refers to a sequence of nucleotides within a nucleic acid molecule that defines the amino acid sequence of an encoded protein. By "isolating DNA" is meant disrupting one or more cells of a library member, e.g., by enzymatic, mechanical, or osmotic means, to release the cellular contents, and removing insoluble materials, lipids, proteins, RNA, and other non-DNA molecules using standard methods or kits available in the art.

"sequencing DNA" refers to determining the nucleic acid sequence of a piece of DNA, such as a gene. Standard methods and commercial services are known in the art. The basic methods for DNA sequencing include the Maxam-Gilbert method and the chain termination method. High throughput techniques have also been developed and are preferably used in the methods of the present technology. These high-throughput techniques include, but are not limited to, Massively Parallel Signature Sequencing (MPSS), Polony sequencing, 454 pyrosequencing, illumina (solexa) sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanosphere sequencing, hellscope single molecule sequencing, single molecule real-time (SMRT) sequencing, and nanopore DNA sequencing.

The methods of the present technology may include additional steps after sequencing the DNA. For example, the DNA sequence of a variant of a parent protein can be compared to the DNA sequence of the parent protein.

The protein of interest may have a desired function selected from the group consisting of enzyme activity, substrate specificity, enzyme stability, and expression titer. The enzymatic activity may include activity at elevated temperatures (e.g., temperatures of 70 ℃, 75 ℃, 80 ℃, or 85 ℃) or at low pH (e.g., pH of 5.5, 5.0, 4.5, or 4.0). The enzyme activity may also include an enzyme activity that is not inhibited by feedback inhibition. Substrate specificity may include specificity for one particular substrate such that no by-products are produced by reaction with other substrates. Enzyme stability may include stability of the enzyme at high temperatures (e.g., temperatures of 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, or 85 ℃), or at low pH (e.g., 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, or 4.0). Expression titer means that the protein of interest is expressed at a high level.

Where it is specified that a method of the present technology includes certain steps "in sequence," other steps may, of course, be included in such a method before, during, or after the listed steps. The order of listing is only in relation to the steps listed in relation to each other and not in relation to any other steps.

II.Method for identifying protein of interest

A. Overview

The present technology relates to the development of a fully automated workflow in a method for screening libraries to identify proteins of interest. The automatic working process of the method of the technology comprises the following steps: (i) automated random pick and dilution (e.g., using an automated liquid handler); (ii) automated plating on plate-sized agar trays (e.g., using a sonic transfer system); and (iii) automated colony picking (e.g., using an automated colony picker). The methods of the present technology provide improvements in cost, efficiency, and user dependence compared to previous workflows. The original workflow required manual random picking (several wells from multiple 384-well plates), manual plating on small agar trays (1 sample per agar plate) and manual colony picking (3 colonies per agar plate, put into 384-well plates). The automated workflow of the present disclosure provides automated random pick and dilution, automated acoustic plating on plate-size agar trays, and trained to identify three different dilutions of the same library member as one area on the agar tray, and to accurately find and pick three colonies thus, the methods of the present technology provide numerous benefits, including:

1. the cost is reduced.

2. The time is reduced.

3. And (3) reducing errors: by eliminating the human variability of manually picking colonies, the requirement for manual intervention (e.g., manual pipetting) is eliminated and accuracy is improved. Colonies were picked and transferred to liquid media (i.e., plate orientation) before being done by hand and were highly variable.

4. The accuracy is improved: acoustic dispensers are a more accurate method of localization that permits the selection of single cell colonies rather than mixed cell populations containing, for example, multiple enzymes, thereby allowing for more accurate gene sequencing. The method of the present technology also provides control over the grid size and dot spacing. For example, there may be 75 spots of 20nL from 3 dilutions at random each time, increasing the likelihood of a single colony.

B. Exemplary embodiments

Various exemplary embodiments of the present technology are described herein. These examples are cited in a non-limiting sense. They are provided to illustrate the broader applicability of the present technology. Various changes may be made in the described techniques, and equivalents may be substituted, without departing from the true spirit and scope of the techniques.

In one aspect, the present disclosure provides a method for identifying a protein of interest, the method comprising screening a library of cells expressing the protein, and then identifying and selecting cells expressing the protein with a desired function. The desired function may be enzyme activity, substrate specificity, expression titer, or any combination thereof.

Another embodiment is a method of identifying an enzyme of interest, said method comprising screening a library of cells expressing the enzyme, and then identifying and selecting cells expressing the enzyme with the desired function; wherein the desired function is enzyme activity, substrate specificity, expression titer, or any combination thereof.

Another embodiment is a method for identifying an enzyme selected from the group consisting of lipase, amylase, xylanase, protease, glucoamylase, glucanase, mannanase, phytase, or cellulase; comprising screening a library of cells expressing the enzyme and then identifying and selecting cells expressing the enzyme with the desired function; wherein the desired function is enzyme activity, substrate specificity, expression titer, or any combination thereof.

The present disclosure also provides a method for identifying a protein of interest, the method comprising: screening a first library of cells expressing proteins; identifying and selecting cells expressing a protein having a desired function; and generating a second library comprising cells expressing proteins with the desired function. Another embodiment is a method for identifying a protein of interest, wherein the second library comprises cells selected from the group consisting of: escherichia coli, Bacillus licheniformis, Bacillus subtilis, Trichoderma reesei, Phaffia foal-shaped yeast, Aspergillus niger, thermophilic bacteria and microzyme. Another embodiment is a method for identifying a protein of interest, wherein the second library comprises cells selected from the group consisting of: escherichia coli, Bacillus licheniformis, Bacillus subtilis, Trichoderma reesei, Phaffia foal-shaped yeast, Aspergillus niger, thermophilic bacteria and yeast; wherein the cell expresses an enzyme and the enzyme is selected from the group consisting of lipase, amylase, xylanase, protease, glucoamylase, glucanase, mannanase, phytase, or cellulase.

Another embodiment is a method for identifying a protein of interest, wherein the microtiter plate comprises wells and the number of wells is selected from the group consisting of: 6. 12, 24, 48, 96, 384 and 1536. Another embodiment is a method for identifying an enzyme of interest, wherein the microtiter plate comprises wells and the number of wells is selected from the group consisting of: 6. 12, 24, 48, 96, 384 and 1536. Another embodiment is a method for identifying an enzyme of interest, wherein the enzyme of interest is a lipase, an amylase, a xylanase, a protease, a glucoamylase, a glucanase, a mannanase, a phytase, or a cellulase; and wherein the microtiter plate comprises wells, and the number of wells is selected from: 6. 12, 24, 48, 96, 384 and 1536.

Another embodiment is a method for identifying a protein of interest, wherein the liquid dilution is a serial dilution, and the serial dilution is repeated 5 times, 4 times, 3 times, and 2 times. Another embodiment is a method for identifying an enzyme of interest, wherein the liquid dilution is a serial dilution and the serial dilution is repeated 5, 4, 3, and 2 times. Another embodiment is a method for identifying an enzyme of interest, wherein the enzyme is a lipase, an amylase, a xylanase, a protease, a glucoamylase, a glucanase, a mannanase, a phytase, or a cellulase and the liquid diluent is a serial diluent; where serial dilutions were repeated 5, 4, 3 and 2 times.

Another embodiment is a method for identifying an enzyme of interest, the method comprising: screening a first library of cells expressing an enzyme; identifying and selecting cells expressing an enzyme having a desired function; generating a second library comprising cells expressing enzymes with the desired function; and arranging the second library into a microtiter plate having a liquid dilution, and the liquid dilution is a liquid comprising a glycerol stock.

Another embodiment is a method for identifying an enzyme of interest, wherein the enzyme is selected from the group consisting of lipase, amylase, xylanase, protease, glucoamylase, glucanase, mannanase, phytase, and cellulase; the method comprises the following steps: screening a first library of cells expressing an enzyme; identifying and selecting cells expressing an enzyme having a desired function; generating a second library comprising cells expressing enzymes with the desired function; aligning the second library into a microtiter plate with a liquid diluent; and then the second library is transferred from the microtiter plate to an agar tray with an acoustic dispenser.

Another embodiment is a method for identifying an enzyme of interest, wherein the enzyme is selected from the group consisting of lipase, amylase, xylanase, protease, glucoamylase, glucanase, mannanase, phytase, and cellulase; the method comprises the following steps: screening a first library of cells expressing an enzyme; identifying and selecting cells expressing an enzyme having a desired function; generating a second library comprising cells expressing enzymes with the desired function; aligning the second library into a microtiter plate with a liquid diluent; and then transferring the second library from the microtiter plate to an agar tray having an acoustic dispenser, wherein the acoustic dispenser transfers 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, 30nL or more of the second library from the microtiter plate to the agar tray.

The disclosure of the present technology also provides a method for identifying a protein of interest, the method comprising: screening a first library of cells expressing proteins; identifying and selecting cells expressing a protein having a desired function; generating a second library comprising cells expressing proteins with desired functions; aligning the second library into a microtiter plate with a liquid diluent; transferring the second library from the microtiter plate to an agar tray with an acoustic dispenser; and incubation and growing colonies on agar trays.

Another embodiment is a method for identifying an enzyme of interest, wherein the enzyme is selected from the group consisting of: lipases, amylases, xylanases, proteases, glucoamylases, glucanases, mannanases, phytases and cellulases, including: screening a first library of cells expressing an enzyme; identifying and selecting cells expressing an enzyme having a desired function; generating a second library comprising cells expressing enzymes with the desired function; aligning the second library into a microtiter plate with a liquid diluent; transferring the second library from the microtiter plate to an agar tray with an acoustic dispenser; and incubating for at least 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 9 hours, 2 hours, 20 hours, 21 hours, 24 hours, hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, 96 hours, 97 hours, 98 hours, 99 hours, 100 hours, 101 hours, 102 hours, 103 hours, 104 hours, 105 hours, 106 hours, 107 hours, 108 hours, 109 hours, 110 hours, 111 hours, 112 hours, 113 hours, 114 hours, 115 hours, 116 hours, 117 hours, 118 hours, 119 hours, and 120 hours; and growing colonies on agar trays.

Another embodiment is a method of identifying an enzyme of interest, comprising: screening a first library of cells expressing an enzyme; identifying and selecting cells expressing an enzyme having a desired function; generating a second library comprising cells expressing enzymes with the desired function; aligning the second library into a microtiter plate with a liquid diluent; transferring the second library from the microtiter plate to an agar tray with an acoustic dispenser; incubating and culturing colonies on agar trays; and picking colonies using a robot.

Another embodiment is a method for identifying an enzyme of interest, wherein the enzyme is selected from the group consisting of: a lipase, an amylase, a xylanase, a protease, a glucoamylase, a glucanase, a mannanase, a phytase, or a cellulase; and the method comprises: screening a first library of cells expressing an enzyme; identifying and selecting cells expressing an enzyme having a desired function; generating a second library comprising cells expressing enzymes with the desired function; aligning the second library into a microtiter plate with a liquid diluent; transferring the second library from the microtiter plate to an agar tray with an acoustic dispenser; incubating and culturing colonies on agar trays; and picking colonies using a robot.

The present disclosure also provides a method for identifying a protein of interest, the method comprising: screening a first library of cells expressing said protein; identifying and selecting cells expressing a protein having a desired function; generating a second library comprising cells expressing proteins with desired functions; aligning the second library into a microtiter plate with a liquid diluent; transferring the second library from the microtiter plate to an agar tray with an acoustic dispenser; incubating and culturing colonies on agar trays; selecting bacterial colonies by using a robot; transferring the colonies to a liquid medium; and growing the culture in a liquid medium and isolating DNA encoding the protein of interest having the desired function.

The present disclosure also provides a method for identifying a protein of interest, the method comprising: screening a first library of cells expressing proteins; identifying and selecting cells expressing a protein having a desired function; generating a second library comprising cells expressing proteins with desired functions; aligning the second library into a microtiter plate with a liquid diluent; transferring the second library from the microtiter plate to an agar tray with an acoustic dispenser; incubating and culturing colonies on agar trays; selecting bacterial colonies by using a robot; transferring the colonies to a liquid medium; growing the culture in a liquid medium and isolating DNA encoding the protein of interest having the desired function; and sequencing the DNA of the target protein.

In another aspect, the present technology provides a method for analyzing a library, the method comprising the following steps in order:

(a) providing a library comprising a plurality of members, wherein each member comprises one or more cells, and wherein each member recombinantly expresses a protein;

(b) arranging the solutions of the members of the library in one or more microtiter plates;

(c) transferring a specified volume of each solution from one or more microtiter plates onto one or more agar trays using an acoustic dispenser;

(d) incubating one or more agar trays until colonies are formed;

(e) picking one or more individual colonies for each member using a robot;

(f) inoculating each selected colony into a liquid culture medium;

(g) incubating the inoculated liquid medium containing the selected colonies, allowing cells present in the selected colonies to multiply;

(h) isolating DNA from the cells in a liquid medium; and the number of the first and second groups,

(i) DNA encoding the protein of interest is sequenced.

In some embodiments, the protein is selected from the group consisting of an enzyme, a peptide, an antibody or antigen-binding fragment thereof, a protein antibiotic, a fusion protein, a vaccine or vaccine-like protein or particle, a factor, a hormone, and a cytokine.

In some embodiments, the enzyme is selected from the group consisting of phytase, protease, beta-glucanase, xylanase, mannanase, lipase, cellulase, glucoamylase, amylase, alpha-amylase, and beta-amylase.

In some embodiments, the one or more cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells. That is, each member of the library to be analyzed comprises one or more prokaryotic or eukaryotic cells. In one such embodiment, the one or more cells are prokaryotic cells and the prokaryotic cells are bacterial cells, i.e., each member of the library to be analyzed comprises one or more bacterial cells. In another such embodiment, the one or more cells are eukaryotic cells, and the eukaryotic cells are selected from the group consisting of fungal cells, yeast cells, animal cells, and plant cells, i.e., each member of the library to be analyzed includes one or more fungal cells, yeast cells, animal cells, or plant cells.

In another embodiment, the one or more cells are selected from the group consisting of escherichia coli cells, bacillus cells, trichoderma cells, coltsia cells, aspergillus cells, thermus cells, and yeast cells.

In some embodiments, each member comprises one or more of an escherichia coli cell, a bacillus cell, a trichoderma cell, a coltsfoot yeast cell, an aspergillus cell, a thermus mold cell, or a yeast cell, wherein each member recombinantly expresses an enzyme selected from the group consisting of phytase, protease, beta-glucanase, xylanase, mannanase, lipase, cellulase, glucoamylase, amylase, alpha-amylase, and beta-amylase.

In some embodiments, each member comprises one or more escherichia coli cells, wherein each member recombinantly expresses an enzyme selected from the group consisting of phytase, protease, beta-glucanase, xylanase, mannanase, lipase, cellulase, glucoamylase, amylase, alpha-amylase, and beta-amylase.

In some embodiments, each member comprises one or more favus foal yeast cells, wherein each member recombinantly expresses an enzyme selected from the group consisting of phytase, protease, beta-glucanase, xylanase, mannanase, lipase, cellulase, glucoamylase, amylase, alpha-amylase, and beta-amylase.

In some embodiments, each member comprises one or more bacillus cells, wherein each member recombinantly expresses an enzyme selected from the group consisting of phytase, protease, beta-glucanase, xylanase, mannanase, lipase, cellulase, glucoamylase, amylase, alpha-amylase, and beta-amylase.

In some embodiments, the protein has a desired function selected from the group consisting of enzyme activity, substrate specificity, enzyme stability, expression titer, and any combination thereof.

In yet another embodiment, the microtiter plate comprises wells such that the alignment of the library member solutions occurs in one or more microtiter plates comprising the wells.

In one or more microtiter plates of any of the methods or embodiments described herein, wherein the number of wells in one or more microtiter plates can be, for example, 6, 12, 24, 48, 96, 384, or 1536.

In one embodiment, in step (b) of any of the methods and embodiments described herein, one or more dilutions of the solution are prepared before aligning the members, optionally wherein the dilutions are serial dilutions. In one embodiment, one or more dilutions are arranged together with undiluted solution. Dilution can be carried out in liquid growth medium, buffer or ultrapure water (e.g., Milli-Q water). Such media, buffers and ultrapure water are known in the art. In one embodiment, one to ten parts of the diluted solution are prepared, preferably two to seven parts of the diluted solution are prepared, and more preferably three to five parts of the diluted solution are prepared. A suitable dilution factor is 1:10, so the first dilution is 1:10, the second dilution is 1:100, the third dilution is 1:1,000, and so on. The dilution preferably results in the formation of a single colony on an agar tray so that individual members can be isolated without contamination by other members.

Thus, the present technology provides a method step (b) comprising preparing a dilution of a solution of the library members and arranging the solution and dilution in one or more microtiter plates optionally comprising wells.

In some embodiments, the dilution is a serial dilution, such that step (b) comprises preparing serial dilutions of the library member solution, and arranging the solution and serial dilutions in one or more microtiter plates optionally comprising wells.

In some embodiments, the solution and/or a dilution of the solution comprises a liquid comprising a glycerol stock. The glycerol stock may comprise, for example, 10% to 60%, preferably 17 to 30% by volume of glycerol.

In some embodiments, the defined volume transferred from the one or more microtiter plates onto the one or more agar trays in step (c) of the methods of the present technology using an acoustic dispenser is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nLnL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30 nL.

In another embodiment, in any of the methods and embodiments described herein, wherein the designated volume transferred is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30nL, the protein has a desired function selected from the group consisting of enzyme activity, substrate specificity, enzyme stability, expression titer, and any combination thereof.

The number of wells in one or more microtiter plates of any of the methods or embodiments described herein, wherein the specified volume transferred in step (c) is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30nL, and wherein one or more microtiter plates can comprise wells of, for example, 6, 12, 24, 48, 96, 384, or 1536.

In one embodiment, in step (b) of any of the methods described herein, wherein the specified volume transferred in step (c) is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30nL, a diluent of the solution is prepared prior to arrangement of the members, optionally wherein the diluent is a continuous diluent. Dilution can be carried out in liquid growth medium, buffer or ultrapure water (e.g., Milli-Q water).

In one embodiment, in any of the methods described herein, wherein the specified volume transferred in step (c) is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30nL, and wherein the dilution or serial dilution is performed in step (b), the one or more cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells. That is, each member of the library to be analyzed comprises one or more prokaryotic or eukaryotic cells. In one such embodiment, the one or more cells are prokaryotic cells and the prokaryotic cells are bacterial cells, i.e., each member of the library to be analyzed comprises one or more bacterial cells. In another such embodiment, the one or more cells are eukaryotic cells, and the eukaryotic cells are selected from the group consisting of fungal cells, yeast cells, animal cells, and plant cells, i.e., each member of the library to be analyzed includes one or more fungal cells, yeast cells, animal cells, or plant cells.

In one embodiment, in any of the methods and embodiments described herein, wherein the specified volume transferred in step (c) is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30nL, and the dilution or series of dilutions are performed in step (b), the protein has a desired function selected from the group consisting of enzyme activity, substrate specificity, enzyme stability, expression titer, and any combination thereof.

In one embodiment, in any of the methods or embodiments described herein, wherein the specified volume transferred in step (c) is 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, 11nL, 12nL, 13nL, 14nL, 15nL, 16nL, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, or 30nL, the solution and/or the dilution of the solution comprises a liquid comprising a glycerol stock solution. The glycerol stock may comprise, for example, 10% to 60%, preferably 17 to 30% by volume of glycerol.

In one embodiment, in step (c) of any of the methods and embodiments described herein, the one or more agar trays are preferably positioned above the one or more microtiter plates, and the one or more agar trays are oriented such that their agar surfaces face the one or more microtiter plates.

In one embodiment, the incubation time in step (d) of any of the methods and embodiments described herein is from 12 hours to 120 hours.

In one embodiment, the incubation temperature in step (d) of any of the methods and embodiments described herein is from 15 ℃ to 50 ℃.

While the technology has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The present technology is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed technology, from a study of the drawings, the disclosure, and the appended claims. The detailed description is merely exemplary in nature and is not intended to limit application and uses.

Examples

The following examples further illustrate the present techniques, however, the scope of the present techniques is not limited thereto. Various changes and modifications may be made by those skilled in the art based on the description of the present technology, and such changes and modifications are also encompassed in the present technology.

Example 1: manual isolation of Individual Pichia pastoris colonies

Three Pichia pastoris strains BD51541, BD51542, and BD51546 containing unique amylase variants were streaked from frozen glycerol stocks to YPD-Zeo¹⁰⁰Agar plates, and cultured at 30 ℃ for three days. Single colonies from each plate were used to load 4mL YPD-Zeo¹⁰⁰The medium was inoculated into 15mL culture tubes. The cultures were grown at 30 ℃ for 18-24 hours with shaking at 250 rpm.

Example 2: universal protocol for automated isolation of individual colonies

Step 1, an ATS source plate is established on an 8-channel Hamilton liquid processor:

-library members are aligned from source glycerol stock plates using a working list

-creating serial dilutions of each library member in ATS compatible 384-flat bottom plates. If a high dilution factor intermediate plate is required, a 96-well plate is used.

Step 2. create a profile file for the ATS run-defining the mapping of the source to the target spot, and the volume of spots to be transferred to the target agar tray. In the software, the target plate was defined as 3456 plate (72 × 48 wells). A grid of this size allows the use of a pattern similar to a 96-well plate, where each well is a variant in a sub-grid spotted on its own in 25 spots (5 x 5). The spot was 20 nL. Plating was then performed in this format to facilitate automated picking on a colony picker, using each of the 96 regions as a picking region.

Step 3. to ensure a single colony, 3 different dilutions of the same library member were spotted side by side. With this arrangement, Omnitray^TMHas a capacity of 32 library members.

Step 4. containing Omnitary^TMThe source plate and target agar are loaded onto the ATS. Using the appropriate atlas file, 6 columns of the source plate are spotted onto one target tray.

Step 5. the agar tray is then capped, placed in an incubator and allowed to grow. The growth conditions, such as temperature and duration, depend on the nature of the library members. Common settings are: for E.coli, overnight at 30 ℃; for Pichia pastoris, 48 hours at 30 ℃.

Exemplary results (E.coli) can be seen in FIG. 2. A close-up of another exemplary result (pichia pastoris) can be seen in fig. 3.

Example 3: automated isolation and sequencing of individual Pichia pastoris colonies

Individual colonies were isolated according to the general protocol of example 2, unless otherwise specified below: using the same three frozen glycerol stocks as in example 1 and an equal mixture of the three cultures obtained in example 1 (generated by normalizing each of the three cultures based on OD600, then combining equal amounts of each culture), one ATS source plate was created on an 8-channel hamilton liquid processor, which was arranged into ATS (EDS biosystems) compatible 384-flat bottom plates (384 Greiner 781090) by using a worklist. The dilution of the serial dilutions by the liquid handler in the same microplate was up to 100,000 times (six total dilutions). A profile file was created according to example 2, but in order to ensure a single colony, all 6 different dilutions (undiluted, 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold) of the four Pichia pastoris were transferred using ATS to the strain containing Omni-Tray^TMYPD-Zeo of target agar trays¹⁰⁰The above. The agar tray was capped, placed in an incubator, and grown at 30 ℃ for 48 hours (see FIG. 4; second plate not drawn, since no colonies grew at higher dilutions).

Example 4: sequencing of genes of interest from individual colonies

Culture PCR for amplifying the full-length amylase gene was performed using each independent culture obtained in example 1 or an equal mixture of each of the three cultures used in example 2 as a template. Colony PCR for amplifying the full-length amylase gene was performed on a plurality of colonies from each dilution group obtained in example 3. By GENEWIZ (New Jersey)State (New Jersey)) sequenced amylase gene products from these PCR reactions and usedThe software performs the analysis.

All three sequences from the cultures derived from single colonies obtained in example 1 showed clean high quality sequencing data of the genes (see figure 5). As expected, sequences from mixed population cultures showed high quality sequences throughout the gene, except for three amino acid residues; 128. 256, and 433 (see fig. 5). These residues are sites in the three pichia strains where the genes differ from each other.

PCR of single colonies from 1-fold, 10-fold, and 100-fold dilution arrays obtained in example 3 showed at least some mixed sequences. All colonies from the 1,000-fold or higher dilution array produced non-mixed sequences, indicating that each colony was a homogenous clonal population. Of the 40 isolated colonies sequenced from the 1,000-fold dilution, each of the three different strains was represented (see FIG. 6).

Example 5: identification of cells expressing proteins with desired Activity

Libraries of cells with different levels of active enzyme expression were prepared and plated as described in examples 2 and 3. These strains were incubated on agar plates containing a substrate on which the expressed enzyme acts and causes a visual change in the appearance of the agar medium. The amylase expressing Bacillus strain showed clear zones on agar plates prepared with 0.1% Red starch after 16 hours incubation at 37 ℃. FIG. 7 shows two libraries with different percentages of active variants. At the lowest dilution rate, a mixed population exists and the entire spot exhibits activity, while at the highest dilution rate, a single colony is obtained and a difference in enzyme activity can be identified.

Equivalents of the formula

While certain embodiments have been illustrated and described, a person of ordinary skill in the art, after reading the above specification, can make changes, substitutions of equivalents, and other types of changes as described herein, to a compound of the present technology, or a salt, pharmaceutical composition, derivative, prodrug, metabolite, tautomer, or racemic mixture thereof. Each of the aspects and embodiments described above may also have incorporated or incorporate such variations or aspects as disclosed in relation to any or all of the other aspects and embodiments.

The present technology is also not limited by the specific aspects described herein, which are intended as single illustrations of individual aspects of the technology. It will be apparent to those skilled in the art that many modifications and variations can be made to the present technology without departing from the spirit and scope of the technology. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Accordingly, it is intended that the specification be considered as exemplary only with the breadth, scope, and spirit of the technology being indicated only by the following claims, definitions therein, and any equivalents thereof.

The embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which are not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be read broadly and not limited. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology.

As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily identified as being sufficiently descriptive and such that the same range is broken down into at least equal two, three, four, five, ten, etc. parts. As a non-limiting example, each range discussed herein may be readily broken down into a lower third, a middle third, an upper third, and so on. As will also be understood by those of skill in the art, all terms such as "highest," "at least," "greater than," "less than," and the like encompass the referenced number and refer to a range that can subsequently be broken down into sub-ranges as discussed above. Finally, as will be understood by those of skill in the art, a range encompasses each individual member.

All publications, patent applications, issued patents, and other documents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. If a definition contained in a text incorporated by reference contradicts a definition in the present disclosure, the contained definition is excluded.

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