A yeast efficient assembly method for large DNA fragments and its application

Abstract

The present invention provides a method for efficiently assembling large-fragment DNA in yeast and its application, and relates to the technical field of molecular biology and synthetic biology. The present invention inserts the MEL1 gene expression cassette of α-galactosidase into the yeast vector, so that the cerevisiae yeast transferred with the expression cassette plasmid can present blue colonies visible to the naked eye on the X-α-Gal plate, which is convenient for distinguishing the correct positive clones and false positive clones in the recombination process, effectively reducing the screening workload, shortening the cycle, and the screening success rate is as high as 92.7%. The blue and white colonies visible to the naked eye of the present invention can be used as the spot picking conditions of the automated equipment, which can improve the efficiency and throughput of the automated screening, and greatly increase the feasibility of the yeast TAR technology in the automated process.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and synthetic biology, and in particular to a yeast efficient assembly method for large-fragment DNA and its application. Background Art

[0002] Gene synthesis of large DNA molecules is of great significance. On the one hand, it can synthesize single structural units so that researchers can determine and optimize the functions of genes. On the other hand, it can also be used to assemble metabolic pathways with economic application value, replace traditional chemical products, reduce carbon emissions, and promote the development of green economy. Today's synthetic biology has entered the era of whole genome synthesis. From the first gene synthesized in 1970 (yeast alanine tRNA gene 77bp) to the synthesis of Escherichia coli genome (4M) in 2020, the length of DNA synthesis has been increasing, realizing the synthesis from single gene to genome, and inspiring humans to challenge the synthesis of more complex higher mammals and even human genomes. Compared with individual genes, the scale of genome synthesis is at the level of hundreds of Kb or even Mb, and the scale of mammalian or human genomes has reached the Gb level. Most commercial gene synthesis is within 10Kb. Large-fragment DNA synthesis technology is one of the key difficulties in the current technology development in the field of gene synthesis. The synthesis, assembly and efficient delivery of large-fragment DNA have become the core foundation of future synthetic genomics.

[0003] At present, in vitro large fragment assembly methods include Gateway, BioBricks, Golden Gate and Gibson Assembly, etc. Among them, standard assembly technologies such as Gateway and BioBricks require the construction of entry vectors, which is time-consuming, costly, and restricted by enzyme cutting sites; Golden Gate technology relies on type II restriction endonucleases and ligases, and is not suitable for the synthesis of DNA in the presence of the above enzymes, and requires analysis of fragment sequences in advance before assembly; traditional Gibson assembly technology is less efficient when assembling multiple fragments, especially for larger fragments, and the assembly success rate is very low. Although the yeast TAR homologous recombination technology assembled in vivo has the advantages of strong assembly ability and strong sequence fidelity, it is the first choice for large-fragment DNA assembly; however, affected by the length of the recombination arm and the number of assembled fragments, the current success rate of yeast TAR homologous recombination technology is low and fluctuates greatly, only 20% to 90%; and traditional TAR recombination can only pick single colonies one by one for PCR identification, which is time-consuming and laborious; in addition, when TAR recombination technology assembles multiple fragments, there are also false positive colonies caused by self-ligation of linearized vectors, which further increases the manpower and cycle during screening. There is no obvious difference between positive clones and false positive clones assembled by conventional TAR on agar plates. Even if an automated spot picking instrument is used, it only increases the screening throughput, but does not improve the efficiency, which is not conducive to the development of high-throughput automated assembly technology. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a yeast efficient assembly method for large DNA fragments and its application, through the interaction between vector expression protein and specific substances in selective culture medium, optimize assembly conditions, make false positive and positive clones show significant phenotypic differences, and increase the positive rate of TAR assembly. Through the above improvements, the workload of manual screening can be reduced, the cycle can be shortened, and screening conditions can be provided for automated spot picking instruments, greatly increasing the efficiency and throughput of automated assembly.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for efficiently assembling large-fragment DNA in yeast, the method comprising the following steps:

[0007] S1, inserting the MEL1 gene expression cassette of α-galactosidase into the yeast vector to obtain the yeast TAR homologous recombination vector;

[0008] S2, linearizing the recombinant vector and transforming it with other DNA fragments to be assembled into Saccharomyces cerevisiae competent cells;

[0009] S3. Spread onto a selective plate containing X-α-Gal, screen based on blue-white spots, and verify by PCR and sequencing to obtain large-fragment DNA.

[0010] MEL1 is a reporter gene of the GAL4 yeast two-hybrid system, encoding a secreted α-galactosidase that can hydrolyze the colorless X-α-Gal substrate and ultimately produce a blue product. Yeast colonies expressing α-galactosidase appear blue on a culture medium containing X-α-Gal, which can visually detect the interaction between endogenous and exogenous proteins in cells.

[0011] Preferably, the MEL1 gene expression cassette includes a Trp expression cassette, a TEF1 expression cassette or a TDH3 expression cassette.

[0012] Preferably, the Trp expression cassette comprises a Trp promoter, a MEL1 gene and an ADH1 terminator; the TEF1 expression cassette comprises a TEF1 promoter, a MEL1 gene and an ADH1 terminator; and the TDH3 expression cassette comprises a TDH3 promoter, a MEL1 gene and an ADH1 terminator.

[0013] Preferably, the nucleotide sequence of the Trp expression cassette is shown as SEQ ID NO.72, the nucleotide sequence of the TEF1 expression cassette is shown as SEQ ID NO.73, and the nucleotide sequence of the TDH3 expression cassette is shown as SEQ ID NO.74.

[0014] Preferably, the yeast vector is pSynoYAC0, pRS416 or pYAC4.

[0015] Preferably, the nucleotide sequence of the yeast vector pSynoYAC0 is shown as SEQ ID NO.108.

[0016] Preferably, Alu sequences of different lengths are added between the start codon and the promoter of the MEL1 gene expression cassette, and the length of the Alu sequence includes 45 / 45 bp, 45 / 65 bp, 45 / 75 bp or 65 / 65 bp.

[0017] Preferably, the MEL1 gene expression cassette includes a TEF1 expression cassette or a TDH3 expression cassette.

[0018] Preferably, the length of the Alu sequence is 65 / 65 bp, and the nucleotide sequence is shown in SEQ ID NO.80.

[0019] The present invention also provides application of the above-mentioned assembly method in efficient assembly of large DNA fragments.

[0020] Beneficial effects of the present invention:

[0021] 1. The present invention inserts the MEL1 gene expression cassette of α-galactosidase into the yeast vector, so that the cerevisiae yeast into which the expression cassette plasmid is transferred can decompose X-α-Gal into a blue substance. Compared with the vector of traditional yeast TAR recombination technology, the present invention significantly improves the screening efficiency, reduces the screening workload, and shortens the cycle.

[0022] 2. The present invention uses TEF1 and TDH3 promoters as promoters of the MEL1 expression cassette, so that the cerevisiae yeast transferred with the expression cassette plasmid can present blue colonies visible to the naked eye on the X-α-Gal plate, and inserts a DNA sequence of up to 130 bp between the promoter and the start codon to reduce false positive white colonies of the vector self-ligation, making it easier to distinguish between correct positive clones and false positive clones in the recombination process, further reducing the manpower and cycle during screening, and the screening success rate is as high as 92.7%.

[0023] 3. Compared with traditional yeast TAR recombination technology, the blue and white colonies visible to the naked eye in the present invention can be used as the picking conditions of automated equipment, which can improve the efficiency and throughput of automated screening, greatly increase the feasibility of applying yeast TAR technology to automated processes, and have great significance for the industrial-scale assembly of large-fragment DNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The agarose gel electrophoresis diagram of the MEL1 gene expression cassette of different promoters in Example 1.

[0025] Figure 2 Schematic diagram of the pSynoYAC0 shuttle vector in Example 1.

[0026] Figure 3 PCR identification results of pSynoYAC0 vector construction of different expression cassettes in Example 1.

[0027] Figure 4 Schematic diagram of comparison of sequencing results of pSynoYAC0 vector construction of different expression cassettes in Example 1.

[0028] Figure 5 (a) is the colony color of the pSynoYAC0 plasmids with different expression cassettes in Example 2 on the X-α-Gal plate; (b) is the product color of the pSynoYAC0 plasmid with the Trp expression cassette in Example 2 in the SC-His culture medium containing X-α-Gal.

[0029] Figure 6 The colony colors of the pSynoYAC0-MEL1 plasmids with different lengths of recombination arms in Example 3 on the X-α-Gal plate.

[0030] Figure 7 This is a schematic diagram of the assembly of the artificial adenovirus genome cloned using yeast TAR homologous recombination technology in Example 4.

[0031] Figure 8 This is a gel image of PCR identification of the artificial adenovirus genome assembled by yeast TAR homologous recombination technology using the shuttle plasmids pSynoYAC0-Alu-130 (a) and pSynoYAC0-MEL1-Alu-130 (b) in Example 4. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0033] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0034] Example 1: Construction of yeast TAR homologous recombination vector

[0035] (1) Obtaining the MEL1 gene expression cassette:

[0036] The DNA sequence of the MEL1 gene was obtained by searching the NCBI database, with the accession number LN997416.1. The MEL1 gene sequence was split into 30 fragments of about 40 to 70 nt in length by sequence software, and single-stranded DNA primers (SEQ ID NO.1 to SEQ ID NO.30) were synthesized by chemical method; then PCR was performed using Phusion high-fidelity DNA polymerase to obtain the DNA fragment of the MEL1 gene (SEQ ID NO.31).

[0037] Using the Saccharomyces cerevisiae genome as a template, the P shown in SEQ ID NO.32 to SEQ ID NO.33 trp -F / P trp -R primer amplified Trp promoter (SEQ ID NO.34); P shown in SEQ ID NO.35 to SEQ ID NO.44 TEF1 -F / P TEF1 -R primer amplified TEF1 promoter (SEQ ID NO.45); P shown in SEQ ID NO.46 to SEQ ID NO.63 TDH3 -F / P TDH3 -R primer amplified TDH3 promoter (SEQ ID NO.64); T shown in SEQ ID NO.65 to SEQ ID NO.70 ADH1 -F / T ADH1 -R primer amplified the ADH1 terminator (SEQ ID NO. 71).

[0038] Using the above-mentioned fragment combination and Phusion high-fidelity DNA polymerase, PCR was performed to amplify MEL1 gene expression cassettes driven by different promoters, namely, Trp expression cassette (Trp promoter + MEL1 + ADH1 terminator) (SEQ ID NO.72), TEF1 expression cassette (TEF1 promoter + MEL1 + ADH1 terminator) (SEQ ID NO.73) and TDH3 expression cassette (TDH3 promoter + MEL1 + ADH1 terminator) (SEQ ID NO.74).

[0039] All the above primers were synthesized by our company, Suzhou Hongxun Biotechnology Co., Ltd., and Phusion high-fidelity DNA polymerase was also prepared by our company. The PCR reaction conditions used were: 98℃ pre-denaturation for 1min; 98℃ denaturation for 15s; 50-60℃ annealing for 30s; 72℃ extension for X s (the specific time depends on the length of the target fragment); 35 cycles; 72℃ extension for another 10min, and storage at 4℃. The agarose gel electrophoresis of the three expression cassettes is shown in the figure below. Figure 1 shown.

[0040] (2) Linearization of shuttle vector pSynoYAC0:

[0041] pSynoYAC0 vector elements such as Figure 2 As shown, the upstream and downstream of the three expression cassettes have 28 bp homologous sequences with the pSynoYAC0 vector. Figure 2 The pSynoYAC0 vector was digested with AscI endonuclease. The 200ul digestion system was as follows: 10ul rCutsmart Buffer, 5ul AscI endonuclease, 20ug plasmid, and the remaining volume was filled with H2O. The above reaction system was placed in a 37°C water bath for 2h of digestion. After digestion, the linearized pSynoYAC0 vector was directly recovered through a nucleic acid purification column.

[0042] (3) Assembly of expression cassette:

[0043] The three expression cassette fragments obtained in step (1) and the linearized vector pSynoYAC0 obtained in step (2) were assembled by Gibson seamless cloning technology. The 20ul reaction system is: 2ul expression cassette fragment, 1ul vector fragment, 7ul H2O, and 10ul Gibson reagent. The above reaction system was placed on a PCR instrument and reacted at 50°C for 45min. After the reaction, the reaction solution was directly added to the E. coli competent cells that had just melted, placed on ice for 1h, heat-shocked at 42°C for 30s, and then ice-bathed again for 10min. Then, 900ul of preheated LB culture medium was added to the competent cells, incubated at 37°C for 1h, and then the bacterial solution was spread on the Cm LB plate and cultured overnight at 37°C.

[0044] (4) Screening of E. coli positive clones:

[0045] A single colony of E. coli was picked from the plate for culture and PCR identification was performed using universal primers M13-47 / M13-48. The empty band was 194 bp, while the correct band size was consistent with the length of the corresponding expression cassette. The gel image of the PCR identification result is shown in the figure below. Figure 3 As shown, the clones with the correct bands were selected for testing, and finally the positive clones with the correct sequence were determined. The sequence comparison results are shown in Figure 4 shown.

[0046] Example 2: Functional verification of shuttle vector pSynoYAC0-MEL1

[0047] In this embodiment, only the pSynoYAC0 shuttle vector is used as an example to verify the function of MEL1, but the actual application is not limited to this vector. The same function can be achieved by using other replicative, episomal and centromere shuttle vectors such as pRS416, pYAC4, etc.

[0048] (1) Preparation of competent cells of Saccharomyces cerevisiae:

[0049] Take the yeast cells frozen at -80℃ and streak them on a sterile YPD plate for activation, and culture them at 30℃ for 36-48h; when a single colony visible to the naked eye appears on the plate, extract the single colony and inoculate it into a sterile liquid YPD medium, and culture it at 30℃ overnight; inoculate the bacterial solution into 50ml fresh YPD medium, and the initial OD600 of the fresh bacterial solution is about 0.4-0.5; place it at 30℃, 220rpm and culture it at a constant temperature until the OD600 of the culture solution reaches 0.9-1.0.

[0050] Transfer the bacterial solution to a centrifuge tube, and collect the yeast cells by centrifugation at 4000rpm for 10min; resuspend and wash the cells with 0.5 times volume (25mL) of sterile water, and centrifuge at 4000rpm for 10min to collect the yeast cells; repeat the above steps once; resuspend the yeast cells with 25mL of 0.1MLiAc and 0.25ml of 1M DTT solution, and culture at 30℃ and 100rpm for 1h; collect the yeast cells by centrifugation at 4000rpm for 10min; resuspend and wash the cells with 25mL of 1M sorbitol solution, and collect the yeast cells by centrifugation at 4000rpm for 10min; discard the supernatant, resuspend the cells with the remaining sorbitol, and dispense the cells into 1.5ml centrifuge tubes with a specification of 100ul / tube, which are yeast competent cells.

[0051] (2) Transformation of Saccharomyces cerevisiae cells with different MEL1 expression cassette plasmids:

[0052] The transformation system was prepared as follows:

[0053] Element Volume(ul) PEG3350 (50%) 520 1MLiAc 72 ssDNA (2mg / ml) 80 Shuttle Plasmid 48(1ug) Total volume 720

[0054] The competent cells were centrifuged at 4000rpm for 2min, and the supernatant was removed; the mixture prepared in the previous step was added, the yeast cells were resuspended by pipetting, and the mixture was mixed by inversion; incubated at 42℃ for 40min, and the mixture was mixed by inversion every 10min; placed on ice for 5min; centrifuged at 4000rpm for 2min, and the supernatant was removed to collect the yeast cells; 6ml YPD culture medium was added and cultured at 30℃ overnight; the yeast cells were centrifuged at 4000rpm for 2min to collect the yeast cells; the cells were resuspended in sterile water and then spread on the SC-His selective plate containing X-α-Gal; the culture was cultured at 30℃ for 48h, and the color of the colonies on the plate was observed.

[0055] The color of the colonies on the plate is Figure 5As shown, according to the colony color, it can be clearly found that the Trp expression cassette plasmid has no obvious blue colony color on the plate, while the TEF1 expression cassette plasmid and the TDH3 expression cassette plasmid have obvious blue colony color on the plate. The monoclonal colony of the Trp expression cassette plasmid was inoculated into the SC-His culture medium containing X-α-Gal, and the cells were collected by centrifugation after 48 hours of culture. The cells were obviously blue when visible to the naked eye. It can be seen that the Trp expression cassette plasmid, the TEF1 expression cassette plasmid and the TDH3 expression cassette plasmid can all make Saccharomyces cerevisiae express MEL1 protein, but the Trp expression cassette is weakly expressed and cannot form blue colonies on the plate containing X-α-Gal, but it can still generate significant blue products when cultured in bacterial liquid.

[0056] Example 3: Optimization of the length of the recombination arm on the shuttle vector pSynoYAC0-MEL1

[0057] TEF1 is one of the strongest yeast constitutive promoters and has a stable expression intensity at different cell stages (Partow S, et al. Yeast (2010) 27 (11): 955-964). Therefore, the present invention's subsequent study selected the TEF1 expression cassette for yeast TAR homologous recombination test. According to reports, after the linearized yeast Yaco vector is transformed into the saccharomyces cerevisiae cell, it is easy to cyclize through the NHEJ (non-homologous end-joining) pathway (Lewis LK, et al. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis (2000) 451 (1-2): 71-89), which is also one of the main reasons for the high false positive of yeast TAR homologous recombination. Because the restriction site EcoRI on pSynoYAC0-MEL1 is located upstream of the ATG start codon of the MEL1 gene, individual base mutations are likely to occur during cyclization, resulting in frameshift mutations and protein inactivation in the MEL1 gene, thereby causing white false positive colonies. To avoid this situation, the present invention adds Alu sequences of different lengths between the promoter and the start codon of the MEL1 gene, and tests its effect on the downstream MEL1 gene. The Alu sequence is the most common moderately repeated sequence in mammalian and human genomes. Since this DNA sequence contains the recognition sequence AGCT of the restriction endonuclease AluI, it is called the Alu sequence.

[0058] (1) Synthesis and assembly of Alu sequences of different lengths:

[0059] Alu sequences of different lengths were synthesized by chemical method, namely 45 / 45bp, 45 / 65bp, 45 / 75bp, 65 / 65bp, 75 / 75bp and 115 / 75bp, and the sequences were SEQ ID NO.77 to SEQ ID NO.82.

[0060] The pSynoYAC0-MEL1 vector was digested with EcoRI, and the Alu sequences of different lengths were assembled into the pSynoYAC0-MEL1 vector by the Gibson method and named Alu-90, Alu-110, Alu-120, Alu-130, Alu-150 and Alu-190, respectively.

[0061] (2) Transformation of Saccharomyces cerevisiae cells with pSynoYAC0-MEL1 plasmids containing Alu sequences of different lengths:

[0062] The pSynoYAC0-MEL1 plasmids with Alu sequences of different lengths were transformed by referring to the transformation steps of Saccharomyces cerevisiae in Example 2; the effect of inserting Alu sequences of different lengths between the promoter and the start codon of the MEL1 gene on the expression of the MEL1 gene was determined based on the color of the colonies of different plasmids on the X-α-Gal plate.

[0063] The color of the colonies on the plate is Figure 6 As shown, it can be clearly found that the plasmids Alu-90, Alu-110, Alu-120 and Alu-130 inserted with shorter Alu sequences have no obvious effect on the expression of the downstream MEL1 gene; while the Alu-150 and Alu-190 plasmid colonies have no visible color reaction on the X-α-Gal plate.

[0064] In this embodiment, only the Alu sequence is used as an example to verify the function of MEL1, but the actual application is not limited to this sequence. The same purpose can be achieved by using other sequences as the spacer sequence between the promoter and the MEL1 gene.

[0065] Example 4: Yeast TAR homologous recombination test of shuttle vector pSynoYAC0-MEL1-Alu-130

[0066] Literature reports that the shortest recombination arm of yeast TAR homologous recombination technology is 60 bp (Noskov VN, et al. Nucleic Acids Research (2001) 29 (6): E32). The subsequent study of the present invention selected the pSynoYAC0-MEL1-Alu-130 shuttle vector for testing.

[0067] In the 1980s, the therapeutic potential of adenovirus as a gene delivery vector was discovered, and it was the first viral gene delivery vector used in humans. The adenovirus genome will not integrate into the host cell, and can be directly translated and transcribed, which is faster than RNA viruses. For cell experiments, the expression peak will appear 36-48 hours after infection; for animal experiments, the expression peak is reached in 3-4 days, and the expression effect can be maintained for about 1-3 weeks. In the past decade, synthetic biology has made significant progress in different fields such as neuroscience, cell therapy and gene therapy, and adenovirus has attracted much attention as a viral vector in gene therapy. Synthetic bioengineering of artificial adenovirus vectors may bring better gene delivery and editing platforms, thereby finding applications in a wide range of therapeutic environments. The present invention uses an artificial adenovirus genome to test the assembly efficiency of pSynoYAC0-MEL1-Alu-130, and the full length of the artificial adenovirus genome is 32kb.

[0068] The adenovirus genome was divided into 9 segments by sequence software, and the sequences were SEQ ID NO.83 to SEQ ID NO.91. Each segment had homologous recombination arms (overlap, OL) of about 60 to 90 bp with adjacent segments at both ends, which were SEQ ID NO.92 to SEQ ID NO.101. First, segments 1 to 9 were split into primers of about 60 nt, and PCR was performed using Phusion high-fidelity DNA polymerase to obtain segments 1 to 9. The PCR reaction system and reaction procedure were the same as in Example 1; then, segments 1 to 9 were assembled into a pUC57 vector by Gibson or restriction ligation, and the sequence correctness was verified by sequencing.

[0069] The plasmids of fragments 1 to 9 were extracted by alkaline hydrolysis, the plasmids were digested by AscI, and fragments 1 to 9 were purified by agarose gel. The digestion recovery method was the same as in Example 2; pSynoYAC0-Alu-130 and pSynoYAC0-MEL1-Alu-130 shuttle vectors were digested by SmaI, and the linearized plasmids were recovered by gel purification. 200 ng of each of the 9 DNA fragments obtained in the above steps were mixed evenly with 100 ng of linearized pSynoYAC0-Alu-130 or pSynoYAC0-MEL1-Alu-130, and the adenovirus genome assembly structure was as shown in FIG. Figure 7 and co-transformed into Saccharomyces cerevisiae cells as shown.

[0070] The transformation steps and system are the same as those in Example 2. After incubation, the competent cells are resuspended in sterile water and the two shuttle vectors are respectively applied to the SC-His selective plate containing X-α-Gal; the plates are cultured at 30°C for 48 hours, and the color of the colonies on the plates is observed. The pSynoYAC0-Alu-130-treated plates are all white colonies, while the pSynoYAC0-MEL1-Alu-130-treated plates have blue and white colonies. 96 white colonies were randomly picked for pSynoYAC0-Alu-130 treatment and pSynoYAC0-MEL1-Alu-130 treatment and inoculated into SC-His culture medium, and cultured at 30°C for 48 hours.

[0071] Multiplex PCR was performed using Taq DNA polymerase to verify the correctness of the adenovirus genome assembly in yeast culture medium. Figure 7 As shown, primers were designed at both ends of OL. The detailed information of primers is shown in Table 1.

[0072] Table 1 PCR primers for identification of artificial adenovirus genome

[0073]

[0074] The 25ul multiplex PCR reaction system is as follows: 2*PCR Mix 10ul, 10pmol / ul primer 0.75ul, bacterial solution 2ul, make up to 25ul with water. The multiplex PCR reaction program is as follows: 98℃ pre-denaturation 1min; 98℃ denaturation 15s; 52℃ annealing 30s; 72℃ extension 30s; 30 cycles; 72℃ extension 5min, store at 4℃. Check the size of the multiplex PCR product by agarose gel electrophoresis, the gel image is as follows Figure 8 As shown. Randomly select the correct positive clones to send for testing 3 reactions, and count the number of clones with correct sequences. The data statistics of yeast TAR homologous recombinant adenovirus genome are shown in Table 2. All the above primers were synthesized by our company, and TaqDNA polymerase was also prepared by our company.

[0075] Table 2 PCR primers for identification of artificial adenovirus genome

[0076]

[0077] The data showed that after only transforming the linearized shuttle plasmid pSynoYAC0-Alu-130 or pSynoYAC0-MEL1-Alu-130 into yeast cells, there were still a considerable number of colonies, which was the false positive caused by the vector self-ligation mentioned above; and the linearized pSynoYAC0-MEL1-Alu-130 also produced 55 white colonies, which may be due to the inactivation of MEL1 caused by the frameshift mutation after mutation. After the adenovirus genome fragments 1 to 9 and the linearized vector were transformed into yeast cells together, the multiple PCR identification results showed that the screening success rate of pSynoYAC0-Alu-130 without the blue-white screening system was only 44.8%, and most of the white colonies were false positives caused by the vector self-ligation; while the screening success rate of white colonies treated with pSynoYAC0-MEL1-Alu-130 with the blue-white screening system was significantly increased to 92.7%. The sequencing results of the 4 positive clones randomly selected by PCR identification were all correct, and it can be judged that the 32kb adenovirus genome was successfully assembled. In this embodiment, the adenovirus genome is only used as a case to verify the high positive rate of the present invention, and the practical application is not limited to this. The present invention can significantly improve the efficiency of assembling all large fragments of DNA (such as natural compound microbial metabolic pathways, microbial genomes, etc.) by yeast TAR homologous recombination technology, which is more efficient, convenient and economical than existing methods, and has higher automation scalability.

[0078] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for efficiently assembling large DNA fragments in yeast, characterized in that: The method comprises the following steps: S1, inserting the MEL1 gene expression cassette of α-galactosidase into the yeast vector to obtain the yeast TAR homologous recombination vector; S2, linearizing the recombinant vector and transforming it with other DNA fragments to be assembled into Saccharomyces cerevisiae competent cells; S3, coating on a selective plate containing X-α-Gal, screening based on blue-white spots, PCR and sequencing verification, and obtaining large DNA fragments; Alu sequences of different lengths are added between the start codon and the promoter of the MEL1 gene expression cassette. The lengths of the Alu sequences include 45 / 45bp, 45 / 65bp, 45 / 75bp or 65 / 65bp. The nucleotide sequences are shown in SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79 and SEQ ID NO.80 respectively.

2. The assembly method according to claim 1, characterized in that: The MEL1 gene expression cassette includes a Trp expression cassette, a TEF1 expression cassette or a TDH3 expression cassette.

3. The assembly method according to claim 2, characterized in that: The Trp expression cassette comprises a Trp promoter, a MEL1 gene and an ADH1 terminator; the TEF1 expression cassette comprises a TEF1 promoter, a MEL1 gene and an ADH1 terminator; and the TDH3 expression cassette comprises a TDH3 promoter, a MEL1 gene and an ADH1 terminator.

4. The assembly method according to claim 3, characterized in that: The nucleotide sequence of the Trp expression cassette is shown in SEQ ID NO.72, the nucleotide sequence of the TEF1 expression cassette is shown in SEQ ID NO.73, and the nucleotide sequence of the TDH3 expression cassette is shown in SEQ ID NO.

74.

5. The assembly method according to claim 1, characterized in that: The yeast vector is pSynoYAC0, pRS416 or pYAC4.

6. The assembly method according to claim 5, characterized in that: The nucleotide sequence of the yeast vector pSynoYAC0 is shown in SEQ ID NO.

108.

7. The assembly method according to claim 1, characterized in that: The length of the Alu sequence is 65 / 65 bp, and the nucleotide sequence is shown in SEQ ID NO.

80.

8. Use of the assembly method according to any one of claims 1 to 7 in the efficient assembly of large DNA fragments.

Images