Rescuing the ompA Deletion Mutant Escherichia coli JW0940 by Reintroducing ompA in the TOPO Cloning Vector pBAD

OmpA is a transmembrane protein that has an essential function in protecting the integrity of the outer membrane of Escherichia coli, and provides a docking point for the donor pilus during conjugation. The plasmid TOPO cloning vector pBAD containing the ompA insert, whose expression is induced by arabinose, was previously designed as a tool to study the relationship between ompA expression and conjugation. The purpose of this study was to introduce the expression system into an ompA -deletion E. coli strain to improve the controlled inducibility of expression of ompA at the outer membrane. The insert orientation in the plasmid was verified through a restriction digest containing EcoRV and HincII. An ompA expression assay resulted in increased ompA expression as increasing arabinose doses were added to the culture medium, and confirmed the presence of OmpA in the outer membrane. Differential ompA expression was not linked with a difference in resistance to sodium dodecyl sulfate. The effects of varying the levels of expression of the ompA gene on the integrity of the outer membrane, and on F conjugation remain yet to be fully elucidated.