Patent ID: 11952596
Assignee: HUBEI UNIVERSITY
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 6:
7. The method according to claim 1, wherein the recombinant strain is obtained by the following steps of
(1) synthesizing the glutamate dehydrogenase gene (gdhA) according to a genomic DNA sequence of Peptostreptococcus asaccharolyticus DSM 20463 to obtain a synthesized gdhA gene, wherein the synthesized gdhA gene has a sequence shown in SEQ ID NO:2, and primers for amplifying the gdhA gene comprise T2-F2 of SEQ ID NO:3 and T2-R2 of SEQ ID NO:4,
amplifying an upstream homologous arm and a downstream homologous arm of a glutamate dehydrogenase gene (rocG) of the Bacillus licheniformis per se through PCR with a genomic DNA of Bacillus licheniformis WX-02 as a template, wherein primers for amplifying the upstream homologous arm comprise T2-F1 of SEQ ID NO: 5 and T2-R1 of SEQ ID NO: 6; and primers for amplifying the downstream homologous arm comprise T2-F3 of SEQ ID NO: 7 and T2-R3 of SEQ ID NO: 8;
(2) linking the upstream homologous arm of the rocG gene, the amplified gdhA gene, and the downstream homologous arm of the rocG gene through overlap-extension PCR to form a target gene fragment in an order of the upstream homologous arm of the gene rocG—the amplified gdhA gene—the downstream homologous arm of the gene rocG;
(3) performing double digestion on the target gene fragment using restriction endonucleases SacI and XbaI to obtain digested target gene fragments, and meanwhile, performing double digestion on a plasmid T2(2)-Ori using the restriction endonucleases SacI and XbaI to obtain linear plasmid fragments;
(4) linking the digested target fragments obtained in step (3) with the linear plasmid fragments obtained in step (3) via T4-DNA ligases, and verifying correctness to obtain plasmids T2(2)-gdhA;
(5) transferring the plasmids T2(2)-gdhA into the Bacillus licheniformis WX-02 to obtain transformants and picking plasmids from the transformants for colony PCR verification to obtain verified strains;
(6) transferring and culturing the transformants obtained in step (5) and performing colony PCR to detect single-exchange strains using T2-KYF of SEQ ID NO: 9 and gdhA-R of SEQ ID NO: 10 as primers; and
(7) inoculating, mixing, and culturing the verified strains obtained in step (5) and the single-exchange strains obtained in step (6) to obtain mixed strains, transferring and culturing the mixed strains and picking transformants in the mixed strains for colony PCR verification to obtain positive transformants, and performing DNA sequencing on the positive transform ants for further verification, thereby obtaining successfully double-exchanged recombinant strains, wherein primers for the colony PCR verification are T2-KYF of SEQ ID NO: 11 and T2-KYR of SEQ ID NO: 12.