Patent ID: 11879124
Assignee: HUBEI UNIVERSITY
Field: Biotechnology (Chemistry)
Classification: CPC C  Y | IPC C

Claim 0:
1. A construction method of a regulation system for gene expression in Zymomonas mobilis, comprising:
constructing a recombinant plasmid for regulating T7 RNAP expression; and
constructing a shuttle plasmid for expressing genes to be driven by a T7 promoter;
wherein the construction method of the recombinant plasmid comprises:
amplifying fragments of T7 RNAP, PBAD, araC, and TetR+Ptet successively;
reversely amplifying a plasmid pZM39 by using primers 39p-F and 39p-R to obtain a vector of pZM39, wherein the primers 39p-F and 39p-R comprise SEQ ID NO.15 and SEQ ID NO.16, respectively;
ligating the vector of pZM39 with the fragments of T7 RNAP, PBAD, araC, TetR+Ptet successively;
transferring ligation product into Escherichia coli DH5α competent cells and selecting positive colonies by using chloramphenicol resistant plates;
verifying to obtain positive transformants by using primers pEZ-dp-F comprising SEQ ID NO.17 that is: ctgaattcgcggccgc and pEZ-15A-R comprising SEQ ID NO.18 that is: cacttcactgacaccctcat; and
cultivating and extracting the positive transformants to obtain the recombinant plasmid;

wherein a nucleotide sequence of the T7 RNAP comprises SEQ ID NO.1;
a nucleotide sequence of the PBAD comprises SEQ ID NO.2;
a nucleotide sequence of the araC comprises SEQ ID NO.3;
a nucleotide sequence of the TetR+Ptet comprises SEQ ID NO.4;
primers for amplifying a fragment of the T7 RNAP are named T7 RNAP-F and T7 RNAP-R, wherein a nucleotide sequence of the T7 RNAP-F comprises SEQ ID NO.7 that is:, atgaacacgattaacatcgctaagaac

 and a nucleotide sequence of the T7 RNAP-R comprises SEQ ID NO.8 that is:, agtagtaggttgaggccgttga;

primers for amplifying a fragment of the PBAD are named PBAD-F and PBAD-R, wherein a nucleotide sequence of the PBAD-F comprises SEQ ID NO.9 that is:, cggccgcttctagagaaaccaattgtccatattgcatcagacattg

 and a nucleotide sequence of the PBAD-R comprises SEQ ID NO.10 that is:, gatgttaatcgtgttcatgggagatcctttctcctctttag;

primers for amplifying a fragment of the araC are named araC-F and araC-R, wherein a nucleotide sequence of the araC-F comprises SEQ ID NO. 11 that is:, atggctgaagcgcaaaatgatcc

 and a nucleotide sequence of the araC-R comprises SEQ ID NO. 12 that is:, ctcgagatctatgggacgttatgacaacttgacggctacatcattc;

 and
primers for amplifying a fragment of TetR+Ptet are named TetR-F and Ptet-R, wherein a nucleotide sequence of the TetR-F comprises SEQ ID NO. 13 that is:, cctcaacctactactttaagacccactttcacatttaagttgtttttct

 

aa

 and a nucleotide sequence of the Ptet-R comprises SEQ ID NO. 14 that is:, ttgcgcttcagccatgggagatcctttctcctctttagatc;

wherein the shuttle plasmid has a replicon derived from Zymomonas mobilis and a replicon derived from Escherichia coli; 
the shuttle plasmid is obtained by replacing f1 origin on plasmid pET22b or pET28a from Escherichia coli with a Zymo-replicon derived from Zymomonas mobilis, and inserting a gene fragment to be driven by the T7 promoter.