CN116426559A

CN116426559A - Method for rapidly synthesizing target fragment from head by using long primer

Info

Publication number: CN116426559A
Application number: CN202310534971.3A
Authority: CN
Inventors: 仲森林; 黄利平; 祁映璇; 管景灏; 姚瑶; 费正月; 刘冠楠
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2023-05-12
Filing date: 2023-05-12
Publication date: 2023-07-14

Abstract

The invention discloses a method for quickly synthesizing a target fragment from scratch by using a long primer, which comprises the following steps: (1) Designing and synthesizing a plurality of long primers with overlapping bases and covering the whole target fragment; (2) Carrying out PCR amplification by using the even number of long primers obtained in the step (1) in an alternating overlapping manner to obtain a PCR product; (3) Directly integrating the PCR product obtained in the step (2) into a linearization vector by utilizing one-step homologous recombination to obtain a recombination product; (4) Transforming the recombinant product obtained in the step (3) into competent cells to obtain a transformant with the target gene; the method takes 2-5 hours from the design of the long primer in the target gene to the final product. The method provided by the invention is suitable for rapid synthesis of long fragment DNA, has the characteristics of short synthesis period and high accuracy, and is suitable for DNA synthesis with high G+C content, repeated sequence or complex secondary structure.

Description

Method for rapidly synthesizing target fragment from head by using long primer

Technical Field

The invention belongs to the field of synthetic biology, and particularly relates to a method for rapidly synthesizing a target fragment from scratch by using a long primer (Rapid DNA synthesis using long primers, RSLP).

Background

Chemical synthesis of DNA sequences provides a powerful tool for efficient expression of genes in heterologous systems and characterization of gene structural functions. However, chemical synthesis makes it possible to generate genes without using template DNA molecules, since template DNA is not always readily available. However, the genetic chemical synthesis method also has many disadvantages including high price, long synthesis period, difficulty in synthesizing DNA having high G+C content, repetitive sequences or complex secondary structure. Therefore, in order to avoid that the experimental period becomes too long due to the synthesis of the target DNA, it is important to develop a simple, rapid and economical synthesis method of DNA.

In recent years, some studies have described methods of oligonucleotide-based DNA sequence synthesis and assembly, such as internal-to-external synthesis (TBIO) based on thermodynamic equilibrium of PCR, two-step gene synthesis based on double-asymmetry PCR ((DA-PCR), overlap extension PCR (OE-PCR), PCR two-step synthetic DNA method (PTDS), and PCR-based precise synthesis (PAS), etc., however, these techniques have significant drawbacks such as relatively high error rate of PCR synthesis, redundancy of PCR system, cumbersome PCR procedure, etc.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a method for quickly synthesizing a target fragment from scratch by using a long primer aiming at the defects of the prior art.

In order to solve the above technical problems, the present invention discloses a method for rapid de novo synthesis of a target fragment using long primers, i.e., an improved version of RSLP of the PTDS method for synthesizing long and accurate DNA sequences. Similar to other DNA PCR synthesis methods, the first step of the RSLP protocol is to design the DNA sequence to be synthesized; codons and g+c content, as well as undesirable restriction enzyme sites or secondary structures, can be optimized in this step; based on the DNA sequence, 80-100 nt oligonucleotides, i.e., long primers, overlapping with adjacent oligonucleotides by about 18-25 bp are designed and chemically synthesized. Next, a second step was performed using PCR using the aforementioned oligonucleotide sets to generate a small fragment of DNA having a length of about 500 to 1000bp (FIG. 1). Here, in order to save time, a plurality of sets of systems (synthesizing different fragments of the same gene) are simultaneously performed (the minimum Tm value is selected as the annealing temperature) at the time of performing the PCR step. Wherein the outermost two primers in each set are used as forward/reverse outer primers, and the remaining largest even number of internal oligonucleotides are used as inner primers; this step should use high fidelity DNA polymerase. And finally, in the third step, a plurality of groups of small fragment DNA (500-1000 bp) are directly assembled on a gene expression vector by means of one-step cloning of an infusion for gene characterization (figure 2).

The method for rapidly synthesizing the target fragment from the head by using the long primer specifically comprises the following steps:

(1) Designing and synthesizing a plurality of long primers with overlapping bases and covering the whole target fragment;

(2) Carrying out PCR amplification by using the even number of long primers obtained in the step (1) in an alternating overlapping way, so as to obtain a PCR product, namely small fragment DNA with the length of about 500-1000 bp;

(3) Directly integrating the PCR product obtained in the step (2) into a linearization vector by utilizing one-step homologous recombination to obtain a recombination product;

(4) Transforming the recombinant product obtained in the step (3) into competent cells to obtain a transformant with the target gene;

the method is carried out from the design of long primers in the target genes to the final products, namely, the total time consumption of the steps (1) - (3) is 2-5 h.

In the above method, if annealing is performed at a temperature of 45℃or higher, the annealing temperature is 45℃or higher in the PCR process, and the annealing temperature is 45℃or higher in the multi-primer PCR process and the vector fragment linearization PCR process.

In the step (1), a proper number of primers is selected according to the designed length of a target fragment, specifically, a fragment of 80-100 nt is selected as a forward outer primer at the 5 'end of the target gene, an overlapping fragment of 18-25 bp long is obtained at the 3' end of the fragment of 80-100 nt, a long primer fragment of 80-100 nt is selected backwards from the (80-100-overlapping fragment) bp position, and the design of the last primer is completed by analogy; the 80 to 100nt, preferably 80 to 90nt, and more preferably 90nt; the 18 to 25bp, preferably 18 to 22bp, and more preferably 20bp.

In the step (1), the overlapping length of the base overlapping is 18 to 25bp, preferably 18 to 22bp, and more preferably 20bp.

In the step (1), the length of the long primer is 80 to 100nt, preferably 80 to 90nt, and more preferably 90nt. An important parameter for DNA synthesis is the length of the oligonucleotides used for PCR. For the RSLP protocol, the present invention uses 90nt oligonucleotides because 90nt provides the best balance between error rate and production cost.

In the step (2), the PCR adopts a template-free mode, wherein two primers at the outermost side when overlapping and alternately extending in the long primers obtained in the even number step (1) are used as forward/reverse outer primers, and the long primer with the largest even number is used as an inner primer.

In the step (2), the PCR system consists of a mixed system of 0.5 mu l of inner primer mixture with a final concentration of 10nM, 1 mu l of forward outer primer with a final concentration of 0.2 mu M, 1 mu l of reverse outer primer with a final concentration of 0.2 mu M, 25 mu l of high-fidelity DNA polymerase, dNTPs and buffer solution, and ddH ₂ O is added to 50 mu l; preferably, the final concentration of 10nM inner primer mixture is obtained by mixing 1. Mu.l of inner primer at a concentration of 10nM, i.e., the inner primers are all obtained by mixing at an equal volume at a concentration of 10 nM.

Wherein the mixed system of the DNA polymerase and the buffer solution is Vazyme P520-01, high-fidelity enzyme can be used in some embodiments, and 2X Phanta Max Master Mix can be used in some embodiments, so that every 100,000bp <1 error can be caused. All previous methods of gene synthesis have errors in the final product, mainly from mutations in the oligonucleotides and mutations introduced during the synthesis of the target DNA sequence mediated by the DNA polymerase.

In the step (3), the linearization vector is obtained by linearizing the vector by using a forward primer and a reverse primer containing homology arms through a PCR technology.

Wherein, the 20bp homologous arm sequences carried on the 5 'end and the 3' end of the vector after linearization of the forward and reverse primers containing homologous arms are respectively identical with the sequences of the 5 'end and the 3' end of the PCR product. If the fragments are inserted into the linearization vector together, the linearization vector has the same sequence at the 5 'end as the 5' end of the large fragment consisting of the plurality of small fragments and the 3 'end as the 3' end of the large fragment consisting of the plurality of small fragments.

In the step (3), the system comprises a linearization vector and n inserts, wherein n is equal to or greater than 5 and equal to or greater than 1, such as n=1, n=2 and n=3.

Wherein the amount of the linearized vector and n inserts is 1:1, and the amount of each linearized vector/insert used is = [0.02×base pair number of linearized vector/insert ] ng, at a final concentration of 0.03 pmol.

In the step (3), the homologous recombination is carried out by the one-step method, the recombination time is 30min, and the recombination temperature is 37 ℃; preferably, the recombinant cells are stored at 4℃after the recombination time has ended.

As can be seen, the RSLP protocol provided by the invention is relatively simple (a plurality of parallel PCRs are cloned on plasmids in one step), rapid (from the design of target genes to the final product-4 h), accurate (less than or equal to 1 error per 100,000bp synthesis) and low in cost.

The beneficial effects are that:

(1) Compared with the scheme similar to the experimental steps of the invention, the invention selects the oligonucleotide chain (long primer) of 90nt, which shortens the synthesis period, reduces the synthesis difficulty, saves the economic cost and provides the best balance between the error rate and the production cost.

(2) The invention adopts a template-free method for synthesizing DNA from the head by utilizing a long oligonucleotide chain (long primer), which is not only less than half of the DNA chain synthesized from the head chemistry in the current market in terms of cost, but also has the advantage of irreplaceability in the aspects of synthesizing the DNA chain with high GC content and complex structure. Most importantly, the invention greatly saves the time for the user to acquire a new gene in terms of time consumption. Under the specific gene segment length, compared with the period of ordering genes in common companies, the time consumption of the invention for successfully constructing the genes is 1/3 times of that of the genes, and with the increase of the gene segment length, the time advantage of the invention is continuously expanded, taking the synthesis of a 10kb DNA chain as an example, the time consumption of the invention is 1/45 times of that of chemical synthesis-! Is very suitable for molecular biology laboratories without templates and in need of new genes.

(3) The method uses homologous recombination to combine multiple gene fragments, and connects multiple DNA fragments with homologous arms, so that the fidelity of the synthesized genes can be further improved, and the time can be saved. Compared with the traditional overlap extension PCR method for connecting a plurality of small fragments by PAS method, the invention adds 20bp homologous arms at two ends of two adjacent DNA fragments directly at the beginning of primer design, and finally clones artificially synthesized genes into a plasmid vector by a one-step cloning mode. Although the fidelity of commercial DNA polymerases is increasing, there is no guarantee that the PCR process will never be erroneous, especially with respect to the principles of the present invention. In this reaction, the number of times of binding of 3' -OH ends of primers by a certain amount of enzymes is 5 times or more that of the template system, and if the prior art PTDS method is adopted, errors are very likely to occur in the process of synthesizing large fragments by using small fragments, which is one of the biggest pain points faced by the PTDS method. The invention can insert the synthesized fragment into any position of the vector by utilizing the one-step cloning technology of the homology arm, and is not limited by the existence of enzyme cutting sites.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 is a demonstration of PCR process.

Fig. 2 is a flowchart illustrating the whole process.

FIG. 3 is a map of pET29a-AMP plasmid.

FIG. 4 is a conceptual diagram of primer design.

FIG. 5 shows the sequencing results (part) of example 1.

FIG. 6 shows the sequencing results (part) of example 2.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

In the following examples, the length of the designed and synthesized long primer was 90nt. Since Jin Weizhi is taken as an example in the market, the primer synthesis charge standard takes 90nt as a watershed, the primer synthesis charge of more than 90nt is 4.2 yuan/nt, and the primer synthesis charge of less than or equal to 90nt is 0.45 yuan/nt. In the synthesis period and the synthesis difficulty, the primer with the synthesis difficulty of more than 90nt is generally higher than the primer with the synthesis difficulty of less than or equal to 90nt, and the synthesis period is also long. In the primer synthesis of 90nt or less, taking the DNA sequence with a 860bp length as an example, 12 primers with 90nt length can be selected, and the total length of the synthesized primers is 1080bp; alternatively, 21 primers 60nt long can be selected, and the total length of the synthesized primers is 1260bp, but the cost of the latter primers is 100 yuan higher than that of the former primers. Therefore, in the primer synthesis of the present invention, 90nt was selected as the sequence length of the long primer designed and synthesized.

The maximum time required for PCR in examples 1 and 2 described below was 1.5 hours, i.e., the PCR was completed within 1.5 hours.

Example 1: construction of pET29a-EI-AMP plasmid

Obtaining a target gene sequence EI [ BBa_K1317003 ], carrying out codon optimization on the target gene sequence EI [ BBa_K1317003 ] to ensure that the target gene sequence EI is suitable for Escherichia coli, and the optimized sequence is shown as SEQ ID NO. 3. The gene of interest was introduced using snapgene software and long primers used for synthesis were designed. Firstly, selecting a 90nt fragment at the 5 'end of a target gene as a forward outer primer, obtaining a 20bp long overlapped fragment at the 3' end of the 90nt fragment, then selecting a 90nt long primer fragment from the (90-overlapped fragment) bp position backwards, and the like until the final primer design is completed. Note that: each primer is designed to extend in the opposite direction to the previous primer. The total number of the designed primers is ensured to be even (the visual display of the primer design is shown in fig. 4). The sequence of the long primer is shown as SEQ ID NO.4-NO. 15. It took 30min.

The system was configured and PCR amplification reactions were performed using the following steps:

1. multi-primer PCR: a855 bp DNA strand was synthesized. Time consuming: 1h5min.

(1) The designed and synthesized 90nt long oligonucleotide primers form 1 group, wherein 12 long oligonucleotide primers (inner primers: SEQ ID NO.5-14, forward outer primers: SEQ ID NO.4, reverse outer primers: SEQ ID NO. 15) contained therein need to synthesize a 855bp DNA fragment. Throughout the process, all reaction solutions should be stored and handled on ice unless otherwise indicated.

(2) 10 inner primers (other primers except the outermost forward/reverse outer primers) were mixed per set: mu.l (10. Mu.M) of 10 inner primers were each placed in a 200. Mu.l PCR tube. All liquids were placed at the bottom of the tube by pipette mixing well and briefly centrifuging with a centrifuge.

(3) 0.5. Mu.l of the inner primer mixture of step (2) was placed in a 200. Mu.l PCR tube, and 1. Mu.l (10 nM) of the forward outer primer and the reverse outer primer were placed in the 200. Mu.l PCR tube, respectively. The final concentration of each inner primer was controlled at 10nM and the final concentration of the outer primer was controlled at 0.2. Mu.M. The multi-primer PCR system is shown in Table 1.

TABLE 1 Multi-primer PCR System setup

(4) PCR procedure shown in Table 2 was used

TABLE 2 Multi-primer PCR procedure set-up

(5) Mu.l of the PCR product was added to 1.0% agarose gel containing 0.1% nucleic acid dye and electrophoresed for 30min at 125V using DL5000 DNA marker (MD 102-01, vazyme) as a reference.

(6) Plasmid pET29a-AMP was derived from laboratory residue and was synthesized by Kirschner Biotech Co., ltd. (FIG. 3), a primer containing homology arms (forward primer nucleotide sequence shown as SEQ ID NO.1, reverse primer nucleotide sequence shown as SEQ ID NO. 2) was designed and linearized by PCR. The PCR system and the procedure are shown in tables 3 and 4.

TABLE 3 vector fragment linearization PCR System

TABLE 4 vector fragment linearization PCR procedure

2. Recovering the target strand DNA. Time consuming: 15min.

(1) The target strip was cut off using a nucleic acid gel cutting apparatus and gel recovery was performed using FastPure Gel DNA Extraction Mini Kit Vazyme.

(2) The gel recovered products were quantified using an ultra-micro ultraviolet spectrophotometer (note that the gel recovered DNA fragment concentration was at least 20 ng/. Mu.l or more).

3. Cloning in one step. Time consuming: 30min.

(1) The one-step cloning system was configured as follows in table 5:

TABLE 5 one-step cloning System

Note that: the molar ratio of linearized vector to insert is 1:1, linearized vector and insert quantization formula: the optimal amount of each linearized vector/insert used = [0.02 x base pair number of linearized vector/insert ] ng at a final concentration of 0.03pmol is important for success or failure of recombination; n=1.

(2) The recombination time is strictly controlled at 30min, the recombination temperature is 37 ℃, and then the recombinant product pET29a-EI-AMP is obtained after the recombinant product is immediately stored at 4 ℃. The recombinant product can be stored at-20deg.C for one week, and thawed when needed.

4. Transformation of recombinant products

(1) Chemically Competent cells (DH 5a component cell, vazyme C502) for cloning were thawed on ice.

(2) 10 μl of the recombinant product was added to 100 μl of competent cells, mixed well with the walls of the flick tube (without shaking, and mixed well), and allowed to stand on ice for 30min.

(3) After heat shock in a water bath at 42 ℃ for 45sec, the mixture is immediately placed on ice for cooling for 2-3min.

(4) 900 μl of SOC or LB medium (without antibiotics) was added, and the 37' C was shaken for 1h (rotation speed 200-250 rpm).

(5) LB plate solid medium containing a final concentration of 100mg/L of kanamycin resistance was pre-heated in an incubator at 37 ℃.

(6) Centrifuge at 5,000rpm (2400 Xg) for 5min, discard 900. Mu.l supernatant. The cells were resuspended in residual SOC or LB medium and gently plated on plates containing the correct resistance with sterile plating bars.

(7) Culturing in an incubator at 37 ℃ for 12-16 hours in an inverted mode.

5. Verification

And picking successfully transformed colonies, verifying by colony PCR, inoculating colony PCR positive transformants, performing amplification culture, preserving, extracting plasmids, and performing PCR verification of plasmids pET29a-EI-AMP. For transformants with positive results of the verification, sequencing was delegated to Suzhou Jinwei Biotech Co. The sequencing result was correct, i.e., confirmed as recombinant bacteria (see FIG. 5 for sequencing results).

Example 2: construction of pET29a-OVA plasmid

Obtaining a target gene sequence OVA [ BBa_K4150006 ], carrying out codon optimization on the target gene sequence OVA [ BBa_K4150006 ] to ensure that the target gene sequence OVA is suitable for Escherichia coli, wherein the optimized sequence is shown as SEQ ID No. 16. Introducing a target gene by using snapgene software and designing a long primer for synthesis, firstly selecting a 90nt fragment at the 5 'end of the target gene as a forward outer primer, obtaining a 20bp long overlapped fragment at the 3' end of the 90nt fragment, then selecting a 90nt long primer fragment from the (90-overlapped fragment) bp position backwards, and the like until the design of the last primer is completed. Note that: each primer is designed to extend in the opposite direction to the previous primer. The total number of the designed primers is ensured to be even (the visual display of the primer design is shown in fig. 4). The sequence of the long primer is shown as SEQ ID NO.17-NO. 34. It took 40min.

1. multi-primer PCR: two DNA strands were synthesized which were 571bp and 677bp in length, respectively. Time consuming: 1.5h.

(1) All designed and synthesized 90nt long oligonucleotide primers are divided into 2 groups with the first group of 8 oligonucleotide primers (inner primers: SEQ ID NO.18-23, forward outer primer: SEQ ID NO.17, reverse outer primer: SEQ ID NO. 24) requiring the synthesis of a 571bp DNA fragment and the second group of 10 oligonucleotide primers (inner primers: SEQ ID NO.26-33, forward outer primer: SEQ ID NO.25, reverse outer primer: SEQ ID NO. 34) requiring the synthesis of a 677bp DNA fragment. Throughout the process, all reaction solutions should be stored and handled on ice unless otherwise indicated.

(2) The inner primers of each set were pre-mixed: two 1. Mu.l (10. Mu.M) inner primers were placed in two 200. Mu.l PCR tubes, respectively. All liquids were placed at the bottom of the tube by pipette mixing well and briefly centrifuging with a centrifuge.

(3) And (2) respectively placing 0.5 mu l of the mixed solution of the two groups of inner primers in the step (2) into 200 mu l of PCR tubes, and respectively placing 1 mu l (10 nM) of each group of corresponding forward outer primers and reverse outer primers into the 200 mu l of PCR tubes. The final concentration of each inner primer was controlled at 10nM and the final concentration of the outer primer was controlled at 0.2. Mu.M. The multi-primer PCR system is shown in Table 6.

TABLE 6 Multi-primer PCR System set-up

(4) PCR procedure shown in Table 7 was used

TABLE 7 Multi-primer PCR procedure set-up

(5) Mu.l of each PCR product was added to 1.0% agarose gel containing 0.1% nucleic acid dye and electrophoresed for 30min at 125V using DL5000 DNA marker (MD 102-01, vazyme) as a reference.

(6) Plasmid pET29a-AMP was derived from laboratory residue and was synthesized by Kirschner Biotech Co., ltd. (FIG. 3), a primer containing a homology arm (forward primer nucleotide sequence shown as SEQ ID NO.35, reverse primer nucleotide sequence shown as SEQ ID NO. 36) was designed and linearized by PCR. The PCR system and the procedure are shown in Table 8 and Table 9.

TABLE 8 vector fragment linearization PCR System

TABLE 9 vector fragment linearization PCR procedure

2. Recovering the target strand DNA. Time consuming: 15min.

4. Cloning in one step. Time consuming: 30min.

(1) The one-step cloning system was configured as follows in table 10:

TABLE 10 one-step cloning System

Note that: the molar ratio of linearized vector to insert is 1:1, linearized vector and insert quantization formula: the optimal amount of each linearized vector/insert used = [0.02 x base pair number of linearized vector/insert ] ng at a final concentration of 0.03pmol is important for success or failure of recombination; n=2.

(2) The recombination time is strictly controlled at 30min, the recombination temperature is 37 ℃, and then the recombinant product pET29a-OVA is obtained after the recombinant product is immediately stored at 4 ℃. The recombinant product can be stored at-20deg.C for one week, and thawed when needed.

4. Transformation of recombinant products

(7) Culturing in an incubator at 37 ℃ for 12-16 hours in an inverted mode.

5. Verification

And picking successfully transformed colonies, verifying by colony PCR, inoculating colony PCR positive transformants, performing amplification culture, preserving, extracting plasmids, and performing PCR verification of plasmids pET29a-OVA. For transformants with positive results of the verification, sequencing was delegated to Suzhou Jinwei Biotech Co. The sequencing result was correct, i.e., confirmed as recombinant bacteria (see FIG. 6 for sequencing results).

The improved method for synthesizing DNA from the head by using the long primer can obtain a plurality of DNA fragments which are designed synthetically with high efficiency and high fidelity by integrating the whole set of experimental procedures, and compared with the conventional DNA synthesis in the market company, the experimental period (from the design of genes to the construction of successfully recombinant plasmids) is reduced by at least 5 days.

The invention provides a method for quickly synthesizing a target fragment from the head by using a long primer, and a method for realizing the technical scheme, wherein the method and the method are a plurality of methods, the method is only a preferred embodiment of the invention, and it is pointed out that a plurality of improvements and modifications can be made by one of ordinary skill in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A method for rapid de novo synthesis of a fragment of interest using a long primer, comprising the steps of:

(1) Designing and synthesizing a long primer with overlapping bases and covering the whole target fragment; the sequence length of the long primer is 80 to 100nt, preferably 80 to 90nt, and more preferably 90nt;

(2) Carrying out PCR amplification by using the even number of long primers obtained in the step (1) in an alternating overlapping manner to obtain a PCR product;

and (3) the total time consumption of the steps (1) to (3) is 2-5 hours.

2. The method of claim 1, wherein the annealing temperature in the method is 45 ℃ or higher.

3. The method according to claim 1, wherein in step (1), the overlap length of the base overlap is 18 to 25bp, preferably 18 to 22bp, further preferably 20bp.

4. The method according to claim 1, wherein in the step (2), the PCR is performed in a template-free mode, wherein the outermost two primers of the long primers obtained in the step (1) are overlapped and alternately extended as forward/reverse outer primers, and the largest even number of long primers are left as inner primers.

5. The method according to claim 4, wherein in step (2), the PCR system comprises a mixed system of 0.5. Mu.l of an inner primer mixture having a final concentration of 10nM, 1. Mu.l of a forward outer primer having a final concentration of 0.2. Mu.M, 1. Mu.l of an reverse outer primer having a final concentration of 0.2. Mu.M, 25. Mu.l of high-fidelity DNA polymerase, dNTPs and buffer, ddH ₂ O is added to 50 mu l; preferably, the final concentration of 10nM inner primer mixture is obtained by mixing the inner primers in equal volumes at a concentration of 10 nM.

6. The method according to claim 1, wherein in step (3), the linearized vector is obtained by linearizing the vector using a primer containing a homology arm by a PCR technique.

7. The method of claim 1, wherein in step (3), the one-step homologous recombination is performed in a system comprising a linearized vector and n inserts, wherein 5.gtoreq.n.gtoreq.1.

8. The method of claim 7, wherein the molar ratio of linearized vector to insert is 1:1 and a final concentration of 0.03pmol of linearized vector/insert per linearized vector/insert is used = [0.02 x base pair number of linearized vector/insert ] ng.

9. The method according to claim 1, wherein in the step (3), the one-step homologous recombination is performed for 30min at a recombination temperature of 37 ℃.

10. The method according to claim 1, wherein in step (3), the recombinant strain is stored at 4℃after the end of the reconstitution time.