A recombinant plasmid pzq12, a recombinant bacterium for synthesizing odd-even number carbon chain monomer copolymerized mcl-pha and its application
A technology for recombining plasmids and plasmids, applied in the field of genetic engineering, can solve the problems of single type of monomer, expensive carbon source, microbial cytotoxicity, etc.
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[0072] The present invention provides a method for preparing recombinant plasmid pZQ12 described in the above scheme, comprising the following steps:
[0073] 1) Using NcoI and BamHI to double-enzyme digest the plasmid pACYCDuet-1 and cimA genes, respectively, to obtain the digested pACYCDuet-1 and cimA genes;
[0074] 2) connecting the digested pACYCDuet-1 and cimA genes in step 1) to obtain the recombinant plasmid pACYCDuet-1-cimA;
[0075] 3) using BamHI and SacI to double-enzyme-digest the recombinant plasmid pACYCDuet-1-cimA and leuBCD gene in the step 2), respectively, to obtain pACYCDuet-1-cimA and leuBCD gene after enzyme digestion;
[0076] 4) connecting the digested pACYCDuet-1-cimA and leuBCD gene in step 3) to obtain the recombinant plasmid pACYCDuet-1-cimA-leuBCD;
[0077] 5) using SacI and AscI to carry out double enzyme digestion on the recombinant plasmid pACYCDuet-1-cimA-leuBCD and ilvA gene of the step 4), respectively, to obtain pACYCDuet-1-cimA-leuBCD and ...
Embodiment 1
[0148] a. extract the total DNA of methane bacteria (Methanobacterium sp.MB1);
[0149] b. Using the total DNA in step a as a template, use primers cimA-F and cimA-R to amplify the cimA gene (the amplification system is 5 μL of 10×HiFi DNA polymerase buffer, 4 μL of dNTPs (2.5 mM), 1 μL of template, cimA -F 1 μL, cimA-R 1 μL, HiFi DNA polymerase 0.5 μL, ddH 2 O 37.5 μL, the total system is 50 μL. The amplification program is: pre-denaturation: 94°C, 5min, 1 cycle; denaturation: 94°C, 45s, annealing: 55-60°C, 45s, extension: 72°C, 2min, in which denaturation, annealing, and extension each undergo 30 cycle; final extension: 72°C, 10 min, to obtain the cimA gene. PCR was used for verification, and the specific results were as follows figure 1 shown. Depend on figure 1 It can be seen that the cimA gene band appears, that is, the cimA gene is obtained.
[0150] (1) Extract the total DNA of Escherichia coli (Escherichia coli MG1655);
[0151] (2) Using the total DNA in step (...
Embodiment 2
[0161] Using plasmid pKD3 or pKD4 as a template, primers ackA-pKD4-F, ackA-pKD4-R, poxB-pKD3-F, poxB-pKD3-R, ldhA-pKD4-F and ldhA-pKD4-R were used to amplify the corresponding Homologous recombination fragments of resistance genes. Electroporation competent cells were prepared, the electroporation process was carried out, and strains after the above-mentioned genes were knocked out were screened to obtain the LZ09 strain. The obtained LZ09 strain was verified by PCR (primers for the PCR verification were ackA-F, ackA-R, poxB-F, poxB-R, ldhA-F and ldhA-R). Specific results such as Figure 4 shown by Figure 4 It can be seen that there are no electrophoresis bands in a, b, and c, indicating that the gene has been knocked out.
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