How to Cite
Abstract
The purpose of this study was to generate an antisense mRNA that can conditionally mimic a catalase knockout phenotype in Escherichia coli. Previous studies revealed that rescue complementation of katE, one of the two catalase encoding genes in E. coli, in the catalase mutant UM2 ( kat E2, kat G15) yielded an unexpected decrease in cell viability. The antisense mRNA method was to provide an alternative approach to control KatE expression by first simulating a double knockout comparable to E. coli UM2. By expressing a katE antisense mRNA under an inducible promoter in the single katG mutant E. coli strain, UM197, the double catalase knockout phenotype can be simulated in the presence of the arabinose inducer, and be rescued upon removal of the inducer. A 760 partial sequence of the endogenous katE gene was amplified from E. coli χ wildtype strain and cloned into pBAD24 vector in an antisense arrangement. The construction and transformation of pBAD24-a katE into UM197 was verified by PCR amplification and was functionally assessed by comparing the relative catalase activity. It was observed that the induced UM197 with the pBAD24 control showed one-fifth the level of catalase activity compared to untransformed UM197 cells in the same media conditions, despite the absence of antisense katE mRNA induction. This suggested that the simulated double knockout might not have been caused solely by the antisense katE mRNA but may also be attributed to effects of the pBAD24 vector. However, consistent results in two catalase assays showed no catalase activity for the induced UM197 pBAD24-a katE. This – – demonstrated that the pBAD24-a katE construct successfully mimicked a KatE KatG double knockout phenotype in the katG knockout UM197 strain which was comparable to the UM2 double knockout positive control strain.