Partial Confirmation of Single katG and katE Knockouts and Double katG/katE Knockouts Created from Isogenic Background of Escherichia coli K-12 Strains

Abstract

Presumptive knockouts of katE and katG catalases were constructed from BW25113 E.coli K-12 strain background via Lambda Red recombination system to generate kat E/ kat G double knockout for a better assessment of the roles of the individual catalases (Narita and Peng, JEMI, 16, 123-8, 2012). The kanamycin resistance cassettes were then removed through FLP-FRT recombination system for consistent antibiotic sensitivity across the laboratory strains. In this study, our goal was to confirm the genotype and phenotype of these knockout strains by PCR, and catalase activity assay with 30% or 2% hydrogen peroxide (H O ). The kat G single knockout and double knockout strains, 2 2 as expected, were catalase positive and negative, respectively. The katE single knockout strain was only catalase positive when the test was done with 2% hydrogen peroxide suggesting a threshold concentration of hydrogen peroxide required for kat G expression. The PCR results confirmed the continued existence of kat E knockout during the process of creating double knockouts. It also identified that the kanR gene insert is present in the presumptive double knockout strain PN11W-4a.