大肠杆菌nmpC基因的快速敲除

生物技术通报 ›› 2013, Vol. 0 ›› Issue (5): 161-164.

大肠杆菌nmpC基因的快速敲除

张丹凤¹, 陈国平², 华秀庭¹, 陈阳¹

(1.漳州师范学院生物科学与技术系，漳州 363000；2.漳州市农业局种植业管理站，漳州 363000)

收稿日期:2013-01-26 修回日期:2013-05-24 出版日期:2013-05-24 发布日期:2013-05-24
作者简介:张丹凤，女，副教授，研究方向：微生物学；E-mail：zh_danfeng@163.com
基金资助:
福建省自然科学基金项目(2010J05088)，漳州市自然科学基金项目(zz2012J05)

Rapid Deletion of nmpC Gene in Escherichia coli Chromosome

Zhang Danfeng¹, Chen Guoping², Hua Xiuting¹, Chen Yang¹

(1. Department of Biological Sciences and Biotechnology，Zhangzhou Normal University，Zhangzhou 363000；2. Planting Management Station，Zhangzhou Agriculture Bureau，Zhangzhou 363000)

Received:2013-01-26 Revised:2013-05-24 Published:2013-05-24 Online:2013-05-24

摘要/Abstract

摘要： 旨在利用Red重组系统和Xer重组系统获得大肠杆菌NmpC外膜蛋白基因删除菌株。采用PCR扩增nmpC基因片段，构建pMD-nmpC载体，EcoR I酶切并经Pfu补平酶切缺口后与difGm片段连接，得到重组质粒T-nmpC∷difGm，PCR扩增获得突变盒nmpC：：difGm，电转化突变盒片段入大肠杆菌细胞，PCR 扩增验证是否获得没有抗性标记的ΔnmpC基因删除菌株。结果显示，成功构建了重组质粒T-nmpC∷difGm，在Red 重组酶和Xer重组酶的介导下，进行同源重组和抗生素标记去除，并经PCR验证得到ΔnmpC基因删除菌株。首次成功获得大肠杆菌ΔnmpC无抗生素标记基因删除菌株，该基因删除菌株可为深入研究NmpC的功能提供研究基础。

关键词: Red重组系统, Xer重组系统, 大肠杆菌, NmpC

Abstract: It was to obtain the nmpC deleted strain of E. coli by using both Red and Xer recombination system. The sequence of nmpC from E. coli K-12 BW25113 was first amplified by PCR then inserted into pMD-18 vector to obtain pMD-nmpC. This vector was then digested by EcoR I and Pfu DNA polymerase was used to create blunt ends after digestion, following ligation with difGm fragment to form T-nmpC∷difGm recombinant. nmpC∷difGm was then amplified by PCR and transformed into E. coli by electroporation, following PCR analysis to confirm the designate ΔnmpC strain of E. coli without antibiotic resistance gene was obtained. Results showed the recombinant vector T-nmpC∷difGm was successfully constructed using a process mediated by Red and Xer recombination system. nmpC gene was deleted by homologous recombination and selectable marker was removed from experiment strain, which was confirmed by PCR. The ΔnmpC strain of E. coli without antibiotic resistance gene was obtained for the first time. This strain would be a promising tool for further investigating the function of NmpC outer membrane protein.

Key words: Red recombination system, Xer recombination system, E. coli, NmpC

张丹凤, 陈国平, 华秀庭, 陈阳. 大肠杆菌nmpC基因的快速敲除[J]. 生物技术通报, 2013, 0(5): 161-164.

Zhang Danfeng, Chen Guoping, Hua Xiuting, Chen Yang. Rapid Deletion of nmpC Gene in Escherichia coli Chromosome[J]. Biotechnology Bulletin, 2013, 0(5): 161-164.

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