Troubleshooting for the Proposed Construction of pBAD24-ompA

Abstract

A better understanding of the mechanisms controlling conjugation efficiency is imperative, as the horizontal transfer of DNA can have profound implications clinically and industrially. OmpA, an abundant outer membrane protein in Escherichia coli, has been demonstrated to have a crucial role during conjugation in gain-of function and lossof-function experiments; however, the effects of varying the abundance of OmpA have not been demonstrated. In this experiment, the construction of a clone containing a pBAD24- ompA construct was attempted. Due to difficulties in the production of the construct, troubleshooting was carried out to determine the source of the quandary. It was hypothesized that the source of the problem could have stemmed from a form of “gene discrimination” of ompA for pBAD24 in either orientation, where the ompA gene preferentially inserted into another vector during ligation.