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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Different responses in mutagenesis induced by quinolone antibacterial agents in two Escherichia coli WP2uvrA/ pKM101
Yayoi Hayasaki1, Sanae Takada, Akiharu Hanamoto
1Medicinal Safety Research Laboratories, Kasai R&D Center, DAIICHI SANKYO Co., LTD. hayasaki.yayoi.xc@daiichisankyo.co.jp
Abstract:
Escherichia coli WP2uvrA/pKM101 strain obtained from a different supplier had a lower response to quinolone antibacterial agents (quinolones) when compared to an ordinary responsive strain. The present study was designed to examine the mechanism of lower susceptibility of the new strain to quinolones. A reverse mutation assay showed the new strain was low responsive to six quinolones compared to a responsive strain, while there was no difference in sensitivity to antibacterial activity of quinolones or the mutagenic activity of the positive control compounds in both strains. The sequence of mucA and B genes, which involve chemical and ultraviolet (UV) -induced mutagenesis through error-prone repair, and the total length of plasmid pKM101 in both strains were identical. Furthermore, transformed WP2uvrA/pKM101 strains, which were made by separately electroporating pKM101 extracted from these two strains into WP2uvrA, showed almost the same responses to the mugatenic- and antibacterial- activity of quinolones as the original responsive strain. The two original strains and the recipient WP2uvrA were proven to have proper genetic characteristics. It was demonstrated that the lower susceptibility of the new WP2uvrA/pKM101 strain to the mutagenesis of quinolones was not due to any changes in either plasmid pKM101 or the characteristics of the host detected by a routine check (tryptophan requirements, sensitivity to UV light and cell membrane permeability) of their genetic characteristics.
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