| CPC C12N 15/70 (2013.01) [C12N 15/66 (2013.01); C12Q 1/686 (2013.01); C12Y 120/01001 (2013.01); C12Y 305/01049 (2013.01)] | 8 Claims |
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1. A method for constructing a recombinant Escherichia coli expression stain which expresses a fusion of a formamidase and a phosphite dehydrogenase, comprising the following steps:
(1) amplifying a formamidase gene fragment with a linker sequence by polymerase chain reaction using a first pair of primers and a genome of Paenibacillus pasadenensis CS0611 as a template to clone a formamidase gene with a linker sequence (for-Linker) of 1041 bp, wherein the first pair of primers comprises a forward primer (primer A1) and a reverse (primer A2), wherein the nucleotide sequence of primer A1 is SEQ ID NO: 4 and the nucleotide sequence of primer A2 is SEQ ID NO: 5, wherein the Paenibacillus pasadenensis CS0611 has China Center for Type Culture Collection (CCTCC) deposit number M 2014458;
(2) ligating the for-Linker into a vector to obtain a recombinant vector comprising the for-Linker (vector-for) and transforming E. coli DH5α competent cells with the recombinant vector for to obtain a recombinant E. coli DH5α containing the recombinant vector-for;
(3) amplifying the for-Linker in the recombinant vector-for by PCR using a second pair of primers and the vector for as a template, wherein the second pair of primers comprises the forward primer A1 and a reverse primer (primer B1), wherein the nucleotide sequence of primer B1 is SEQ ID NO: 6;
(4) amplifying a phosphite dehydrogenase gene fragment by PCR using a third pair of primers and a genome of Klebsiella pneumoniae OU7 as a template to clone a phosphite dehydrogenase gene (ptx) of 1008 bp, wherein the third pair of primers comprises a forward primer (primer B2) and a reverse primer (primer B3), wherein the nucleotide sequence of primer B2 is SEQ ID NO: 7 and the nucleotide sequence of primer B3 is SEQ ID NO: 8, wherein the Klebsiella pneumoniae OU7 has CCTCC deposit number M 2017449;
(5) generating a fusion gene (for-Linker-ptx) by overlapping PCR amplification using a fourth pair of primers and the amplified for-Linker of step (3) and the amplified ptx of step (4) as templates, wherein the fourth pair of primers comprises the forward primer A1 and the reverse primer B3;
(6) digesting the for-Linker-ptx fusion gene with BamHI and EcoRI and ligating the digested for-Linker-ptx fusion gene into a pGEX-2T expression plasmid digested with BamHI and EcoRI to obtain a recombinant plasmid comprising the for-Linker-ptx fusion gene (pGEX-for-Linker-ptx), and transforming E. coli DH5α competent cells with the recombinant plasmid pGEX-for-Linker-ptx to obtain a recombinant E. coli DH5α comprising the recombinant plasmid pGEX-for-Linker-ptx;
(7) extracting the recombinant plasmid pGEX-for-Linker-ptx from the recombinant E. coli DH5α comprising the recombinant plasmid pGEX-for-Linker-ptx and transforming E. coli BL21(DE3) competent cells with the recombinant plasmid pGEX-for-Linker-ptx to obtain a recombinant E. coli expression strain; and
(8) inoculating a LB medium with the recombinant E. coli expression strain, inducing expression of the for-Linker-ptx fusion gene, collecting the recombinant E. coli expression strain, adding the recombinant E. coli expression strain to a MOPS medium comprising formamide and phosphite, and culturing the recombinant E. coli expression strain in the MOPS medium comprising formamide and phosphite.
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