The lac Promoter May Not Regulate brkA Expression in Plasmid pDO6935 in Escherichia coli

Abstract

BrkA is an autotransporter protein found in Bordetella pertussis that is regulated by the Bordetella virulence gene (Bvg) system. It has been researched in pDO6935-transformed Escherichia coli, with previous research suggesting that the presence of a lac promoter is necessary for BrkA expression in these models. lacI^Q is a constitutively expressed gene found in pHN678 that encodes the LacI repressor, which when in the absence of lactose or analogs, should bind to the operator downstream of the lac promoter, thereby preventing downstream gene expression. Therefore, the objective of this study was to manipulate this mechanism by introducing lacI^Qinto a pDO6935-derivative, pENS, to create an IPTG-inducible BrkA expression system in E. coli cells, assuming that the lac promoter was responsible for BrkA expression. We created pTEAM, a construct containing lacI^Q downstream of BrkA on the pDO6935-derived pENS plasmid, which was transformed into UT5600 E. coli cells prior to visualization for BrkA expression. Contrary to our hypothesis, our data showed that BrkA was still being expressed with strong signals by our pTEAM construct in the absence of IPTG whereas our pENS constructs expressed little to no BrkA. This result suggests that the lac promoter may not regulate BrkA expression in pDO6935.