Rapid Deletion of nmpC Gene in Escherichia coli Chromosome

Abstract

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

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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