Determining the Relationship Between the lac Operon and the Expression of brkA on pDO6935 in Escherichia coli Using a Promoterless Green Fluorescent Protein Reporter Gene

Abstract

The lac operon regulatory system in Escherichia coli plays a role in controlling gene expression through transcriptional regulation. Understanding the regulation of specific genes is essential for elucidating their roles in bacterial physiology and pathogenesis. We chose to investigate green fluorescent protein (GFP) as a proxy for BrkA, an autotransporter protein in Bordetella pertussis responsible for serum resistance and host cell adherence. In this study, we aimed to introduce an additional copy of lacI into the system to establish a tighter regulation, as well as deleting the promoter region of the lac operon of E. coli to investigate its effects on downstream GFP expression as a proxy for brkA expression. Isopropyl β-d-thiogalactoside (IPTG)-controlled GFP expression was demonstrated as an indicator of operon activity, and an increased GFP expression was observed upon IPTG induction, indicating a tighter regulation of GFP, further reinforcing the regulatory role of the lac operon on the downstream expression of GFP. Due to time restraints, the deletion of lac promoter region did not work out as planned, suggesting a room for us to reflect and troubleshoot. Our findings highlight the importance of the lac operon and its utility in studying BrkA expression in E. coli.