[Show abstract][Hide abstract] ABSTRACT: A new technique for conducting site-directed mutagenesis was developed. This method allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of ß-galactosidase. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. In this paper, 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 knock-out ompR gene was constructed through overlapping PCR, digested by Not and 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.