利用Red重组系统敲除大肠杆菌O157∶H7的waaL基因

Knockout of waaL Gene of Escherichia coli O157∶H7 by Red Recombination System

目的:利用λ噬菌体Red重组系统敲除大肠杆菌O157∶H7的waaL基因。方法:以pKD4为模板扩增出与waaL基因上下游同源的、含有卡那霉素抗性基因的PCR产物。然后电击转化到大肠杆菌O157∶H7中,利用Red重组系统,通过卡那霉素抗性基因两侧的waaL基因序列在体内与waaL基因发生同源重组,置换了O157∶H7基因组中的waaL基因。并进一步利用卡那霉素抗性基因两侧的FRT位点,通过FLP位点专一性重组将卡那霉素抗性基因敲除。结果:成功构建了敲除waaL基因且不带卡那霉素抗性基因的菌株。

Objective： To construct a waaL knockout mutant of Escherichia coli O157∶H7.Methods： Using plasmid pKD4 as a template,the kanamycin-resistant gene flanked by homologues of waaL gene was amplified by PCR.The PCR products were electro-transferred into the O157∶H7,with the help of the Red recombinant system,WaaL gene were replaced by the kanamycin-resistant gene.Then the kanamycin-resistant gene was eliminated by FLP-promoted recombination system.Result： waaL gene of the E.coli O157∶H7 was completly eliminated and the kanamycin-resistant gene was also eliminated.