An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

Abstract:

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

• Periodical: Applied Mechanics and Materials (Volumes 195-196)
• Pages: 407-411
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.195-196.407
• Online since: August 2012
• Authors: Mu Qing Qiu
• Keywords: Gene Replacement, ompR Gene, Overlapping PCR, Site-Directed Mutagenesis

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