Construction and Application of Inducible Yeast Transformation and Recombination System
A technology of recombinant vector and construction method, which is applied in the field of Saccharomyces cerevisiae, can solve the problem of low TAR cloning efficiency, achieve the effect of improving transformation efficiency and reducing pollution
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Embodiment 1
[0036] Embodiment 1: Construction of iTAR vector
[0037] 1. The construction of recombinant vector pSH-SceI comprises the following steps:
[0038] (1) Primer design: artificially design and synthesize specific primer sequences.
[0039] SceIFor:5'-cgatatcaagcttatggaattgtgagcggataacaattcatgcatatgaaaaacatcaa-3';
[0040] SceIRev:5'-catccttagcgccgtaaatcaatttatttcaggaaagtttcgg-3';
[0041] (2) PCR amplification: The configuration of the PCR reaction system is shown in Table 1.
[0042] Reagent name stock solution concentration Volume added to PCR reaction system 5×Q5Reaction Buffer 5× 10μL 5×Q5 High GC Enhancer 5× 10μL dNTPs 10mmol / L 1μL Forward Primer 10μmol / L 2.5μL Reverse Primer 10μmol / L 2.5μL Q5 High-Fidelity DNA Polymerase 5U / μL 0.5μL DNA template 50ng / μL 2μL wxya 2 o
ddH 2 O to a final volume of 50 μL
[0043] The PCR program was pre-denaturation at 98°C for 30s; denaturation...
Embodiment 2
[0079] Example 2: An iTAR vector pSHTB10-HR12 captures hyg-containing R The application of elements to large fragments of DNA comprises the following steps:
[0080] 1. Recombinant strain E.coli BL21-hyg R build
[0081] (1) Primer design:
[0082] TE7For:5'-cagtgctgacaatacaaccgttcaggctgtagcaagtacgagcttgctcctgatc-3';
[0083] TE7Rev: 5'-gtatccagcagcctgattcggctgagtgaacatcagttattcctttgccctcggac-3';
[0084] P1: 5'-gagcgtcaacttaaagctg-3';
[0085] P2: 5'-gaagtggtacggcgatgcgc-3';
[0086] P3:5'-gtcgatgcgacgcaatcgtc-3';
[0087] P4: 5'-gtccgactctaagatgtcac-3';
[0088] (2) PCR amplification: The PCR reaction system is 10 μL of 5×Q5 PCR buffer, 10 μL of 5×enhancer, 1 μL of dNTPs (10 mmol / L), 2.5 μL of each primer (10 μmol / L), 2 μL of plasmid DNA (about 50 ng / μL), Q5 DNA polymerase (5U / μL) 0.5 μL, add ddH 2 0 to 50 μL. The PCR program was pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C, 30 cycles; and finally exte...
Embodiment 3
[0099] Example 3: The application of an iTAR vector pSHTB10-HR34 in capturing polymyxin gene clusters, including the following steps:
[0100] (1) Minigene synthesis:
[0101] S3: 5'-gactgacactgataatacgactcactataggatatctaaaacgtgcgccggttttagagctagaaatagcaagtt-3';
[0102] S4: 5'-gactgacactgataatacgactcactataggcttttaccgatgaagagagggttttagagctagaaatagcaagtt-3';
[0103] (2) Genome digestion of Paenibacillus polymyxa SQR-21: DNA fragments S3 and S4 were transcribed in vitro, and gRNA-3 and gRNA-4 were recovered. Mix SQR-21 genomic DNA with Cas9 enzyme, gRNA-3 and gRNA-4, the enzyme digestion system is SQR-21 genomic DNA (about 25μg / μL) 20μL, Cas9 enzyme (70pmol) 1μL, gRNA-1 (5μg) 1μL , gRNA-2 (5 μg) 1 μL, Cas9 buffer 4 μL, add ddH 2 0 to 40 μL, 37 ° C overnight enzyme digestion, recovery of digestion products.
[0104] (3) Polymyxin gene cluster capture: Transform the purified enzyme-cleaved product (1 μg) into BY4742 / pSHTB10-HR34 competent cells induced by galactose, spread SG...
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