Genetic Compensation of E. coli C156 with pBAD-ompA for the Investigation of Conjugation Efficiency

Abstract

OmpA is a membrane protein known to serve a stabilizing function in mating aggregates of Escherichia coli. The disruption of OmpA function has been shown to reduce conjugation efficiency. Currently, little progress has been made in creating a reliable construct that allows ompA expression to be differentially controlled or over expressed. Such a construct would allow researchers to further elucidate the precise role of ompA expression in F-conjugation. To this effort, the current study generated a pBAD TOPOompA construct using the TOPO TA cloning – technology in an attempt to transform into an ompA E. coli C156 strain. The presence and orientation of pBAD TOPOompA was verified using PCR screening with novel pBAD TOPO primers as well as Hinc II restriction digest. Secondary transformation of the new construct into E. coli C156 was performed, and pBAD clones were isolated. A 10 kb band of undigested plasmid shown in the plasmid preparation of a potential transformant supports the presence of pBAD TOPOompA in the E. coli C156 clone.