Construction of pBAD-clones using the TOPO TA Cloning System

Previous studies have shown that conjugation efficiency decreases when the outer membrane protein, OmpA, was inactivated in Escherichia coli. To study the role of modulated OmpA expression in conjugation, construction of a pBAD24- ompA clone that would allow controlled gene expression under the regulation of arabinose-inducible promoter was attempted in the past but had no success. To assess whether problems arose due to the cloning design or other intrinsic difficulties of cloning ompA, the current investigation attempted to use an alternate TOPO TA cloning system to construct pBADompA. Primers were designed to amplify ompA and restriction analysis verified that the PCR product was consistent with ompA ’s expected banding pattern. Transformation results showed successful recovery of several clones carrying pBAD constructs. Construct pKT10W4 had a higher molecular weight which suggested that it carried a large insert worth further investigation. Due to time constraints, the identity and directionality of the insert had yet to be confirmed as ompA. Since no successful pBADompA clones with correct orientation have been confirmed, results remained inconclusive as to whether any intrinsic difficulties associated with cloning ompA exist.