Plasmid library construction method based on Red / ET recombinant engineering and application thereof

Abstract

The invention belongs to the technical field of gene cloning and assembling, and particularly relates to a plasmid library construction method based on Red / ET recombination engineering and application of the plasmid library construction method. According to the invention, an appropriate linear expression vector is constructed, and 50bp short sequences at two ends of the vector are used as homologous arms. The two homologous arms are introduced to the two ends of all DNA sequences to be studied, so that the two homologous arms can be subjected to homologous recombination with the vector, the cotransformation linearization vector and the target DNA sequence are integrated into escherichia coli with sufficient RecE and RecT expression, and plasmid libraries carrying different target DNAs can be constructed through one-step line-line recombination. On the basis, homologous arms of A-B or B-A versions are introduced to the two ends of a plurality of genes with a synergistic effect relationship, and co-expression plasmids in different combination modes can be constructed by one-step line-line recombination, so that the synergistic effect between the genes is conveniently explored or interaction proteins are determined, and the method has a good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of gene cloning and assembly, and specifically relates to a plasmid library construction method based on Red / ET recombination engineering and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Synthetic biology aims to design and reconstruct biological systems through metabolic engineering strategies of design-build-test-learn. These designs need to rely on appropriate tools to be built and implemented. Standardized biological components are the toolbox of synthetic biology. Therefore, the acquisition, characterization and sharing of biological components are a necessary and important part of the development of synthetic biology.

[0004] Due to the lag of efficient characterization methods, the number of biological components currently characterized is far less than the actual demand, and due to the incompleteness of reported characterization work, more than 200 of the more than 20,000 biological components included in the Registry of Standard Biological Parts (http: / / parts.igem.org / Main_Page) established by the Massachusetts Institute of Technology have failed. Therefore, the establishment of efficient component characterization and standardized methods is necessary to ensure the quantity and quality of the excavated biological components.

[0005] The mining and characterization of biological elements usually rely on flux DNA cloning technology, manipulating DNA with undefined functions to be connected to vectors for expression and functional testing. However, the overall progress of high-throughput cloning technology is slow. At present, in the characterization of biological element sets, the construction of plasmid libraries mainly relies on the sequential action of restriction endonucleases and ligases, including Biobrick and Bglbrick, which are developed based on the combined use of type Ι restriction endonucleases, and NOMAD, which is developed based on the type Ⅱ S restriction endonucleases with relatively separated recognition sites and cleavage sites. With the expansion of the construction scale, this method is limited by the available restriction sites on the one hand, and the cost of introducing restriction sites and using commercial enzymes is increased. In addition, this method will also introduce a foreign sequence before the gene sequence, which may affect the function of the DNA sequence under study. Other flux processing methods, such as the site-specific recombinase-based scheme, only have the ability to transfer gene set flux, and still cannot do without the construction of plasmids one by one. The library construction method that combines restriction enzyme cutting and homologous recombination is still limited by the number of restriction sites.

[0006] Red / ET recombineering is a genetic manipulation technology that does not rely on enzyme cleavage and ligation. This technology does not require the use of commercial enzyme preparations. The phage-derived recombinase mediates homologous recombination between DNA molecules in bacteria. Redα and RecE are exonucleases that start cutting from the 5' end of double-stranded DNA to form local single-stranded DNA, exposing the homologous sequence at the 3' end. Redβ or RecT is a single-stranded annealing protein that is responsible for protecting the single strand and mediating annealing between homologous sequences. RecE and RecT can mediate linear recombination between linear DNA, and Redα and Redβ mediate linear-loop recombination between linear DNA and circular DNA molecules. The efficiency of linear recombination is higher than that of linear-loop recombination. In addition, only about 50bp of homologous arms are needed for recombination to occur, which can be easily introduced to both ends of the target gene through primers. These characteristics make it have the potential to develop into an efficient library construction method. Summary of the invention

[0007] In view of the problems existing in the above-mentioned prior art, the present invention provides a plasmid library construction method based on Red / ET recombination engineering and its application. Specifically, the present invention designs and constructs a suitable linearized expression vector according to the host strain and the research content, and uses the 50bp short sequence at both ends of the vector as a homology arm. The two homology arms are introduced to both ends of all DNA sequences to be studied so that they can undergo homologous recombination with this vector, and the linearized vector and the target DNA sequence set are co-transformed into Escherichia coli with sufficient RecE and RecT expression, so that a one-step line-line recombination construction can be achieved to obtain a plasmid library carrying different target DNAs. The above method shortens the time used for characterization of the DNA sequence set, and is convenient for exchanging or sharing biological elements to be characterized between different strains, without the need to reconstruct the DNA sequence set, and only the expression vector needs to be changed. On this basis, by introducing two versions of homology arms, AB or BA, at both ends of multiple genes with a synergistic relationship, a one-step line-line recombination can be constructed to obtain co-expression plasmids in different combinations, so as to facilitate the exploration of synergy between genes or the determination of interacting proteins. Based on the above research results, the present invention is completed.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a method for constructing a plasmid library based on Red / ET recombination engineering, wherein the construction method is completed by co-transforming a mixture of a DNA sequence set and a linearized expression vector into a host cell, wherein both ends of each sequence in the DNA sequence set carry homologous sequences required for recombination with the linearized expression vector; thereby each DNA sequence can be constructed by homologous recombination with the linearized expression vector in the host cell to obtain a circular plasmid.

[0010] The length of the homologous sequence is about 50 bp, so as to facilitate homologous recombination based on Red / ET technology.

[0011] Based on the above method, the second aspect of the present invention provides the application of the above method in constructing a plasmid for co-expressing multiple genes in different combinations in one recombination.

[0012] The application is completed in the above-mentioned host cell. Specifically, the linearized expression vector and the genes carrying the adapter homology arms are arranged in a certain order under the mediation of the recombinase, and co-expression plasmids with different combinations are obtained by recombination construction between homologous sequences.

[0013] The third aspect of the present invention provides a kit, which may include the DNA sequence set and linearized expression vector in the above method, and a host cell.

[0014] A fourth aspect of the present invention provides the use of the above method, application, and kit in any one or more of the following:

[0015] (a) Gene interaction studies;

[0016] (b) Protein-protein interaction studies;

[0017] (c) Construction of genetically engineered strains.

[0018] The beneficial technical effects of one or more of the above technical solutions are:

[0019] The above technical solution uses engineered enzymes expressed in E. coli instead of commercial enzyme preparations, and relies on homologous recombination between sequences instead of restriction enzyme ligation which is often limited by enzyme cutting sites. Under the premise of controllable costs, a plasmid library containing 80% of the substrates of the provided DNA set can be constructed at one time. And through the design of homologous arms on both wings of the gene, different combinations of genes can be constructed in one recombination to obtain multiple co-expression plasmids.

[0020] The above technical solution reduces the workload of the library construction process and shortens the working time required for flux characterization of functional elements. In addition, the function of constructing a combinatorial expression plasmid makes it more convenient to identify certain gene combinations that require specific genes to cooperate with each other to perform their functions. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1The present invention is used to construct a library based on the Red / ET recombination engineering library construction method for characterizing bacterial promoters.

[0023] Figure 2 This is the plasmid construction map used to build a library to characterize the endogenous promoter of Escherichia coli Nissle 1917. The candidate promoter is placed upstream of the fluorescent reporter gene egfp, and the terminator upstream of the promoter is used to block the interference of the upstream gene on the transcription of egfp, and the fluorescence intensity indicates the strength of the promoter.

[0024] Figure 3 The Venn diagram was used to select genes with high transcription levels in three periods to determine candidate promoter sequences.

[0025] Figure 4 This is a diagram showing the working mechanism of the Red / ET recombination engineering library construction method, using the construction of a bacterial endogenous promoter library as an example.

[0026] Figure 5 It is the fluorescence value distribution diagram of the promoter plasmid transformant population detected by flow cytometry.

[0027] Figure 6 The richness of promoter plasmids obtained by one-time recombination construction by the method established by the present invention is shown. km and gentamicin resistance gene promoter P genta Except for 1, the other 41 promoters were obtained by one recombination screening.

[0028] Figure 7 It is a schematic diagram of the process of constructing a combination expression plasmid of multiple heat-resistant related proteins through the method established by the present invention.

[0029] Figure 8 The invention is a mutant strain of Geobacillus stearothermophilus with improved heat resistance, which is obtained by screening after transformation with a heat resistance-related protein combination expression plasmid. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0032] In a typical embodiment of the present invention, a method for constructing a plasmid library based on Red / ET recombination engineering is provided, wherein the construction method is completed by co-transforming a mixture of a DNA sequence set and a linearized expression vector into a host cell, wherein both ends of each sequence in the DNA sequence set carry homologous sequences required for recombination with the linearized expression vector; thereby, each DNA sequence can be constructed by homologous recombination with the linearized expression vector in the host cell to obtain a circular plasmid.

[0033] The length of the homologous sequence is about 50 bp, so as to facilitate homologous recombination based on Red / ET technology.

[0034] The host cell is a bacterial host cell with Red / ET recombination engineering established. Specifically, the bacterial host cell can at least express RecE and RecT. In the present invention, the bacterial host cell can be Escherichia coli, and can further be GB2005-dir or its derivative strains GB2005-dir-pir and GB2005-dir-gyrA462 whose genome is integrated with recE and recT genes derived from Rac prophage.

[0035] The DNA sequence set is determined by the research purpose, and the types of replicons and markers in the linearized vector are determined according to the research purpose and the characteristics of the host bacteria, and are not specifically limited here.

[0036] In particular, at the DNA collection acquisition stage, a 50 bp long sequence, called a homology arm, is introduced at both ends of each sequence. The sequences are homologous sequences upstream and downstream of the linearized vector, respectively, and the same homology arms are carried at both ends of all sequences in the DNA sequence collection. After each sequence in the DNA sequence collection is mixed in equal amounts and transformed into a host cell (such as Escherichia coli capable of expressing RecE and RecT) in the form of a mixture for linear recombination, the recombinant population is cultured and the plasmid is extracted to obtain a plasmid library.

[0037] It can be seen from the above technical solution that what is actually provided is a one-step recombination throughput method for cloning a DNA sequence into a vector to construct a plasmid library.

[0038] Based on the above method, in another specific embodiment of the present invention, there is provided application of the above method in constructing a plasmid for co-expression of multiple genes in different combinations in one recombination.

[0039] The application is completed in the above-mentioned host cell. Specifically, the linearized expression vector and the genes carrying the adapter homology arms are arranged in a certain order under the mediation of the recombinase, and co-expression plasmids with different combinations are obtained by recombination construction between homologous sequences.

[0040] In particular, in the process of preparing the "promoter-target gene" expression module, each gene will be prepared as an expression module carrying two versions of homologous arms, AB or BA, so that the expression modules of different genes can be recombined into the vector together in a certain order to construct co-expression plasmids of different combinations.

[0041] In another specific embodiment of the present invention, a kit is provided, which may include the DNA sequence set and linearized expression vector in the above method, and a host cell.

[0042] In another specific embodiment of the present invention, the above method, application, and kit are provided for use in any one or more of the following:

[0043] (a) Gene interaction studies;

[0044] (b) Protein-protein interaction studies;

[0045] (c) Construction of genetically engineered strains.

[0046] The following examples are used to describe in detail the Red / ET recombination engineering-based library construction method of the present invention. The following examples are only for explaining the present invention and are not intended to limit the content thereof.

[0047] Unless otherwise specified, the reagents and biological materials used in the present invention are all commercial products. Various molecular biological operations not described in detail are all general operation methods.

[0048] Example 1: Construction of an endogenous promoter library of E. coli Nissle 1917 to screen for strong promoters

[0049] Escherichia coli Nissle 1917 is a wild strain preserved in the laboratory, referred to as EcN. The scheme for constructing its endogenous promoter library and characterizing promoters is as follows: Figure 1As shown, the transcriptome of the plant at different growth stages was measured and the genes with high transcription intensity in each stage were obtained by analysis. The promoter regions of these genes were cut to construct a plasmid library, which was transformed into the native bacteria to detect activity.

[0050] 1. Preparation of expression vectors required for library construction

[0051] like Figure 2 As shown, in this example, pBBR1-km, which works stably in E. coli, is used as an expression vector for constructing a promoter library, a fluorescent reporter gene egfp is introduced to indicate the strength of the promoter, and a terminator is introduced downstream of the Km resistance gene in the vector by PCR to avoid vector elements interfering with the transcription of the reporter gene. The vector used to introduce the terminator is pBBR1-Km-1 / Terminator-pBBR1-Km-2.

[0052] In order to improve the efficiency of homologous recombination in the process of constructing a plasmid library, in this embodiment, the part of the plasmid except the promoter, that is, the linearized vector into which the terminator has been introduced by PCR, and the fluorescent reporter gene are fused into a DNA fragment by overlap PCR. The 3' end of egfp has been introduced in advance by GFP-in-1 / pBBR1-GFP-2 primers to introduce a 30bp homology arm for overlapping with the linearized pBBR1-Km-terminator vector. The primer pair used for preparing the linearized expression vector pBBR1-Km-terminator-eGFP for constructing the promoter library by overlap PCR is GFP-in-1 / Terminator-pBBR1-Km-2.

[0053] 2. Determination and acquisition of candidate promoters

[0054] In this embodiment, candidate promoters are determined based on the transcriptome of the strain at different growth stages. The specific scheme is as follows:

[0055] (1) Transcriptome determination: EcN cells grown to the mid-logarithmic growth period, the early stationary period, and the mid-stationary period were cultured and collected, and after quick freezing in liquid nitrogen, the transcriptome was determined by Beijing Novogene Technology Co., Ltd.

[0056] (2) Determination of candidate sequences: The measured transcriptome data were preprocessed, and the genes were sorted from high to low according to the FPKM value in each period. The top 200 genes in the FPKM value were taken as one set of data, so each strain had three sets of data. The intersection of the three sets of data for the same strain was taken using the Draw venn Diagram online webpage. The results are as follows: Figure 3As shown, the genes in the intersection were ranked according to the sum of the FPKM values ​​of the genes in the three periods, and the top 50 genes were selected for research. The DNA sequence between the start codon of each gene and the stop codon of its upstream gene was intercepted as the promoter of the gene, and a total of 50 promoter candidate sequences were obtained, which were named promoters NP1 to NP50.

[0057] (3) The EcN bacteria were used as templates to amplify the promoter candidate sequences NP1-NP50 with the linearized expression vector pBBR1-Km-terminator-eGFP homology arms. The primer pairs involved were referenced to Sequence Table 1. The specific operation method of colony PCR was to add 50 μL of sterile deionized water to a 1.5 mL EP tube, pick a single colony on the plate and pipette it into the water to mix it, pierce the EP tube cover and place it in a microwave oven to boil for 10 minutes, and use the boiled bacterial solution as a template in a conventional PCR system to obtain each candidate promoter.

[0058] 3. Construction of Promoter Library

[0059] The promoter plasmid library of the present invention is constructed by co-transforming all promoter DNA and vector into Red / ET recombinant engineering bacteria Escherichia coli GB2005-dir (Escherichia coli Red / ET engineering bacteria preserved in this laboratory) and then undergoing one in vivo recombination. The working principle is as follows: Figure 4 The specific plan is as follows:

[0060] (1) All promoter sequences were mixed in equal proportions and then mixed with the vector in a ratio of 2:1. The mixture was spotted onto a Millipore desalting membrane and desalted for 30 min before use.

[0061] (2) Pick a single colony from the E. coli GB2005-dir culture dish and inoculate it into 1.3 mL of antibiotic-free LB medium. Culture at 960 rpm and 37°C for about 10 h.

[0062] (3) Transfer 50 μL of the above bacterial solution to 1.3 mL of antibiotic-free LB medium and culture for 2 h until OD 600 Close to 0.6.

[0063] (4) Add 35 μL of arabinose (working concentration 1400 μg mL) -1 ) and then continue to culture for 40 minutes to induce the expression of sufficient amounts of RecE, RecT and Redγ in GB2005-dir.

[0064] (5) After the induction culture was completed, the cells were collected by centrifugation at 9600 rpm for 1 min and the supernatant was discarded.

[0065] (6) Resuspend the cells in 1 mL of sterile deionized water and centrifuge at 9600 rpm for 1 min to collect the cells; repeat this step twice.

[0066] (7) Resuspend the bacteria at the bottom of the centrifuge tube with 30 μL of sterile deionized water, add the desalted DNA mixture from (1) and mix by pipetting. Transfer to a dry, sterile 1 mm electroporation cup and place in an electroporator for 1350 V electroporation.

[0067] (8) Add 1 mL of antibiotic-free LB medium and the bacterial solution to the electroporation cup and mix well. Transfer to a new 1.5 mL centrifuge tube and resuscitate at 37°C and 960 rpm for 2 h.

[0068] (9) After recovery, centrifuge at 9600 rpm for 30 seconds, discard the supernatant, retain about 100 μL of culture medium to resuspend the bacteria, spread on LB plates containing corresponding antibiotics, and invert and culture in a 37°C incubator for 12 hours.

[0069] (10) Use an inoculation loop to scrape all the colonies growing on the resistance plate into a 250-mL Erlenmeyer flask containing 50 mL of resistance LB liquid culture medium, place it in a shaking cabinet at 37°C and 200 rpm for 6 h, and use the Tiangen plasmid extraction kit to extract the plasmid to obtain the promoter plasmid library.

[0070] 4. Characterization of bacterial endogenous promoters

[0071] In this example, the characterization of bacterial endogenous promoters was completed by transforming the promoter plasmid library constructed in Escherichia coli into the native bacterium Escherichia coli Nissle 1917 (EcN). km -eGFP-Km EcN and carrying pBBR1-P genta -eGFP-KmEcN was used as a control because the kanamycin resistance gene promoter P km and gentamicin resistance gene promoter P genta It is generally considered to be a strong promoter of Escherichia coli. The specific scheme is as follows:

[0072] (1) Promoter library transformation into EcN: Culture a single EcN colony in 1.8 mL of antibiotic-free LB medium at 960 rpm and 37°C for 10 h. Then take 50 μL of the bacterial solution and transfer it to OD 600 Close to 0.6. Collect the cells by centrifugation at 9600rpm for 1min and wash them twice with 1mL of sterile deionized water. Resuspend the cells at the bottom of the centrifuge tube with 30μL of sterile deionized water, add 1μg of the desalted promoter plasmid library, mix well by pipetting, transfer to a dry, sterile 1mm electric shock cup, place in an electroporator for 1350V electric shock treatment, and then resuscitate with 1mL of antibiotic-free LB medium for 2h. After the recovery, centrifuge at 9600rpm for 30s to concentrate the bacterial solution to 100μL and apply to a 20μg·mL -1LB plates containing Km antibiotics were inverted and placed in a 37°C incubator for 12 h;

[0073] (2) Analysis of the richness of transformants by flow cytometry: All colonies were scraped from the resistance plate into 1.8 mL of resistance LB liquid medium, cultured at 37°C and 960 rpm until the mid-logarithmic phase, centrifuged at 9600 rpm for 1 min to collect the cells, then washed twice with 1 mL of ice-cold PBS buffer, and finally resuspended in 1 mL of PBS buffer. After appropriate dilution, the cells were loaded onto a BD FACSAria Fusion flow cytometer. The results are shown in Figure 2. Figure 5 As shown, under the same cell type and living state (SSC / FSC), the cells in the control group were in the low fluorescence brightness range (~10 1 ), while the fluorescence brightness among transformants varied, ranging from 0.5×10 1 -0.5×10 5 This indicates that the transformants are rich in variety and the library construction method based on Red / ET recombination engineering can achieve the goal of obtaining multiple plasmids through one transformation.

[0074] (3) 0.5×10 3 and 1×10 4 As the boundary, the transformant population was divided into three gates: P1 with weak fluorescence, P2 with medium-intensity fluorescence, and P3 with strong fluorescence. The sorting function of BD FACSAria Fusion flow cytometer was used to collect 10,000 bacteria from each gate, coated on resistant LB plates, and cultured under appropriate conditions.

[0075] (4) Then, in an ultra-clean workbench, colonies with different fluorescence brightness were selected from the plate with the help of a blue light instrument and streaked on the plate. The colonies were used as templates for amplification with primers pBBR1-pro-check-1 / 2, and the products were sequenced to determine the sequence of the promoter on the plasmid. A total of 41 transformants of promoter plasmids were obtained.

[0076] (5) The above transformants were inoculated into a 96-well plate and cultured in a microplate shaker at 37°C and 960 rpm. 150 μL of bacterial solution was sampled into the 96-well plate at the middle of the logarithmic phase, the early stage of the stable phase, the middle of the stable phase, and the decay phase. The OD was measured using a Tecan M200pro microplate reader. 600 And fluorescence value, according to the formula RFU = (sample fluorescence value - culture medium fluorescence value) / (sample OD 600 - Culture medium OD 600 ) to calculate the relative fluorescence intensity of each sample. Each sample was repeated 3 times to complete the characterization of 41 endogenous promoters of EcN. The results are shown in Figure 6 As shown, a promoter Pgenta The endogenous promoter NP42 of P. genta In addition, 15 promoters such as NP17, NP3, and NP32 were obtained that were superior to kanamycin resistance gene promoter P km Among them, NP2, NP3, NP6, NP17, NP26, NP30 and NP32 also showed high activity and good stability in the middle and late growth stages. The above promoters have the potential to be used in the engineering transformation of E. coli Nissle 1917.

[0077] Example 2: Construction of a random combination expression plasmid for heat resistance-related proteins to improve the heat resistance of thermophilic Bacillus

[0078] The Geobacillus stearothermophilus SL-1 involved in this example is an oil reservoir native microorganism separated by the inventor in a certain block of Shengli Oilfield in the early stage, and the Escherichia coli E. coli GB2005-dir involved is the Escherichia coli Red / ET engineering bacteria preserved in this laboratory.

[0079] 1. Construction of random combination expression plasmids for heat resistance-related proteins

[0080] In this example, the pBBR1-Km plasmid was used as a vector for expressing the above-mentioned protein, and the plasmid pBBR1-Rha-Redγ-Redαβ-Km preserved in this experiment was used as a template. The pBBR1-Km-1 / 3 primer was used to amplify the pBBR1-Km linearized vector, and the 50 bp sequence at its 5' end was used as the homology arm A, and the 50 bp sequence at its 3' end was used as the homology arm B.

[0081] The genome sequence of Geobacillus stearothermophilus SL-1 was analyzed and it was found that it encodes a variety of proteins related to bacterial thermotolerance, including MurD, CysM, DnaK, DnaJ, HtpX, GroES, GroEL, HslO, GrpE, Hsp20, Hsp33, HemW, ClpB, and ClpX. (1) Using boiled bacterial solution as a template, murD, cysM, dnaK, dnaJ, htpX, groES, groEL, hslO, grpE, hsp20, hsp33, hemW, clpB, and clpX genes were amplified by PCR. In this process, a primer for P-terminal binding was introduced into the 5' end of each gene. km The 30 bp sequence of the promoter overlaps with each gene. km Promoter overlap gets "P km-gene" fragments. Using these gene modules as templates, AP with different versions of homology arms, AB and BA, were prepared by PCR. km -gene-B and BP km -gene-A gene expression module, please refer to Sequence Table 2 for specific primer combinations.

[0082] E. coli GB2005-dir was inoculated into 1.3 mL of antibiotic-free LB medium and cultured at 960 rpm and 37 °C for 10 h. Then 30 μL of the bacterial solution was transferred to culture until OD 600 When the pH value was close to 0.4, 35 μl of arabinose was added and cultured for 40 min to induce sufficient expression of RecE, RecT and Redγ. After centrifugation at 9600 rpm for 1 min, the cells were collected and washed twice with 1 mL of sterile deionized water, and finally resuspended with 30 μL of sterile deionized water. km -gene-B and BP km -gene-A fragments were mixed in equal proportions, 2 μg of the mixture was mixed with 1 μg of the linearized vector pBBR1-Km, and then added to the resuspended bacterial solution after desalting. After 1350V electric shock treatment, it was transformed into GB2005-dir intracellular random combination co-expression plasmids, such as Figure 7 As shown, AP with AB version homology arms km -gene-B can be recombined with pBBR1-Km vector alone to form a circular plasmid, or it can be combined with any number of amorphous gene modules with homology arm sequence of "BA-AB" ("BP km -gene-A+AP km -gene-B") are pulled by homology arms and connected to the pBBR1-Km vector in sequence. In short, the resulting plasmid always carries an odd number of genes.

[0083] The electroporated bacterial solution was revived and cultured for 2 h in 1 mL of LB medium without antibiotics. After the recovery, the bacterial solution was concentrated by centrifugation to 100 μL and coated with 20 μg mL -1 The LB plate containing Km antibiotic was inverted and cultured in a 37°C incubator for 12 h. All colonies on the plate were scraped and cultured and plasmids were extracted to obtain a mixture of expression plasmids of different types of heat-resistant related gene combinations.

[0084] 2. Plasmid transformation host improves the heat resistance of strains

[0085] The obtained combined expression plasmid mixture was transformed into Geobacillus stearothermophilus SL-1 to screen for mutants with improved heat resistance. The specific transformation method is as follows:

[0086] (1) Bacillus stearothermophilus SL-1 was cultured at 65°C until OD 600 Value is approximately 1.0.

[0087] (2) The cells were collected by centrifugation at 8000 rpm for 1 min at 4°C, washed three times with sterile ice water as buffer at 4°C, and then concentrated in 50 μl of sterile water to prepare competent cells.

[0088] (3) Add 1 μg of plasmid to 50 μl of competent cells and mix well. Transfer to a dry and sterile 2 mm electroporation cuvette and place in a Bio-Rad electroporator for electroporation at 2500 V, 200 Ω, and 25 μF.

[0089] (4) Resuspend the bacterial solution in 1 mL of recovery medium, incubate at 48°C for 2 h, spread on a resistant plate and incubate at 65°C for 16 h.

[0090] A single colony on the resistant plate was picked and inoculated into 1.8 mL of resistant LB medium, and cultured at 80°C and 960 rpm to screen for transformants of Geobacillus stearothermophilus with improved heat resistance. Figure 8 As shown, 4 mutant strains capable of growing at 80°C were screened.

[0091] Table 1 Oligonucleotide sequences used in Example 1

[0092]

[0093]

[0094] Table 2 Summary of oligonucleotide sequences used in Example 1

[0095]

[0096]

[0097] Table 3 Summary of oligonucleotide sequences used in Example 1

[0098]

[0099]

[0100] Table 4 Summary of oligonucleotide sequences used in Example 1

[0101]

[0102] Table 5 Summary of oligonucleotide sequences used in Example 1

[0103]

[0104] Table 6 Summary of oligonucleotide sequences used in Example 1

[0105]

[0106]

[0107] Table 7 Summary of oligonucleotide sequences used in Example 1

[0108]

[0109]

[0110] Table 8 Summary of oligonucleotide sequences used in Example 2

[0111]

[0112]

[0113] Table 9 Summary of oligonucleotide sequences used in Example 2

[0114]

[0115]

[0116] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A method for constructing a plasmid library based on Red / ET recombination engineering, characterized in that: The construction method is completed by co-transforming a mixture of a DNA sequence set and a linearized expression vector into a host cell, wherein both ends of each sequence in the DNA sequence set carry homologous sequences required for recombination with the linearized expression vector; thereby each DNA sequence can be constructed by homologous recombination with the linearized expression vector in the host cell to obtain a circular plasmid.

2. The construction method according to claim 1, characterized in that: The length of the homologous sequence is 50 bp.

3. The construction method according to claim 1, characterized in that: The host cell is a bacterial host cell with Red / ET recombination engineering established. Specifically, the bacterial host cell can at least express RecE and RecT.

4. The construction method according to claim 3, characterized in that: The bacterial host cell is Escherichia coli, and can further be GB2005-dir or its derivative strains GB2005-dir-pir and GB2005-dir-gyrA462, the genome of which is integrated with recE and recT genes derived from Rac prophage.

5. The construction method according to claim 1, characterized in that: All sequences in the DNA sequence set carry the same homologous sequence at both ends; each sequence in the DNA sequence set is mixed in equal amounts and transformed into a host cell in the form of a mixture with a linearized vector for linear recombination, and the recombinant population is cultured and the plasmid is extracted to obtain a plasmid library.

6. Application of the construction method according to any one of claims 1 to 5 in the construction of a plasmid for co-expression of multiple genes in different combinations in one recombination.

7. The use according to claim 6, characterized in that The application is completed in the host cell. Specifically, the linearized expression vector and the genes carrying the adapter homology arms are arranged in a certain order under the mediation of the recombinase, and co-expression plasmids with different combinations are obtained by recombination construction between homologous sequences.

8. The use according to claim 7, characterized in that In the process of preparing the "promoter-target gene" expression module, each gene will be prepared as an expression module carrying two versions of homology arms, AB or BA, so that the expression modules of different genes can be recombined into the vector together in a certain order to construct co-expression plasmids of different combinations.

9. A kit, characterized in that: The kit comprises the DNA sequence set and linearized expression vector in the construction method according to any one of claims 1 to 5, and a host cell.

10. Use of the construction method according to any one of claims 1 to 5, the application according to any one of claims 6 to 8, or the kit according to claim 9 in any one or more of the following: (a) Gene interaction studies; (b) Protein-protein interaction studies; (c) Construction of genetically engineered strains.

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