Development of a high-efficient scarless genetic modification method for Yersinia pestis
10.7644/j.issn.1674-9960.2017.03.011
- VernacularTitle:鼠疫耶尔森菌基因组无痕修饰方法的建立
- Author:
Lisheng XIAO
;
Zhizhen QI
;
Ruichen Lü
;
Kai SONG
;
Rong CHEN
;
Min WANG
;
Hailian WU
;
Haihong ZHAO
;
Yajun SONG
- Keywords:
Yersinia pestis;
homologous recombination;
genome modification
- From:
Military Medical Sciences
2017;41(3):209-212,221
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes.Methods The resistance fragment, including upstream and downstream homologous arms of targeted regions, was reamplified by asymmetric PCR.The amplicons were introduced into Y.pestis harboring plasmid pKD46.With the induction of L-arabinose,the recombinant related enzymes: Exo, Beta and Gam, were expressed to guide the homologous recombination.A donor plasmid, pKSI-1, which carried the desired modification fragment flanking by I-SceⅠ recognition sites, was introduced into Y.pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains:△waaA and waaA(△9nt), were successfully constructed for Y.pestis strain 201.Scarless modification introduces no extra modification to the genome, and it is ideal for comprehensive functional genomic studies.
