Using the GFP Reporter Plasmid pEAH23A-1Δ to Investigate the lac Operator Region Driving brkA Expression from Plasmid pDO6935 in E. coli

Abstract

The lac operon is a well-studied, genetic regulatory system capable of tightly modulating the transcription of associated genes in Escherichia coli, principally controlled by a lac promoter that is repressed by a proximal lac operator. Previous research has demonstrated lac-mediated regulation of virulence factor BrkA, a Bordetella pertussis-derived Type V autotransporter, through novel plasmid constructs harbouring brkA downstream of the lac promoter and operator. This putative brkA promoter region was studied in pEAH23A to identify the determinants of BrkA expression via GFP fluorescence, for which the lac promoter region was deemed necessary and sufficient. However, this finding has yet to be validated, and the role of the lac operator in driving BrkA expression remains understudied. As such, we attempted to disrupt the lac operator in pEAH23A through site-directed mutagenesis, creating pEAH23A-1Δ, and assessed BrkA expression within E. coli DH5ɑ and BL21 DE3 transformants using GFP fluorescence imaging and a microplate assay as a proxy. Given its canonically repressive role, we hypothesized that disruption of the lac operator would increase GFP expression, and thus BrkA expression. While this increase was observed in pEAH23A-1Δ, our mutagenesis was imprecise with potentially confounding consequences. This observation suggests that the lac operator may be implicated in the repression of brkA, necessitating further exploration.