利用Red重组系统敲除大肠杆菌 O157:H7的waaL 基因

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中国生物工程杂志 ›› 2011, Vol. 31 ›› Issue (10) : 83-87.

技术与方法

利用Red重组系统敲除大肠杆菌 O157:H7的waaL 基因

杨公进, 马中瑞, 信国, 陈敏

作者信息 +

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

YANG Gong-jin, MA Zhong-rui, XIN Guo, CHEN Min

Author information +

文章历史 +

摘要

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。

Abstract

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.

导出引用

杨公进, 马中瑞, 信国, 陈敏. 利用Red重组系统敲除大肠杆菌 O157:H7的waaL 基因[J]. 中国生物工程杂志, 2011, 31(10): 83-87

YANG Gong-jin, MA Zhong-rui, XIN Guo, CHEN Min. Knockout of waaL Gene of Escherichia coli O157:H7 by Red Recombination System[J]. China Biotechnology, 2011, 31(10): 83-87

中图分类号： Q789

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