The Distance of the lac Regulatory Region to the Translation Start Codon of gfp on Plasmid pEAH23A Does not Change GFP Expression Levels

Résumé

Bordetella pertussis, the causative agent of whooping cough, uses various strategies to evade host immune defenses, notably through its autotransporter protein Bordetella resistance to killing (BrkA) which blocks complement-mediated killing. Therefore, understanding how brkA expression is regulated is crucial for understanding its role in pathogenesis. Previous research has demonstrated the involvement of the lac regulatory region, located upstream of the brkA locus on the pDO6935 plasmid in brkA expression. When this region was cloned onto a promoterless plasmid, it was able to drive GFP expression. However, the impact of the proximity of regulatory regions on gene expression remains poorly understood. In this study, we investigated how the distance between the lac promoter and a reporter gene, gfp, on a plasmid, impacts GFP expression levels. Utilizing site-directed mutagenesis, plasmids pBRAT24C and pBRAT24D were created, with shorter distances between the lac promoter and gfp relative to pEAH23A. Plasmids were transformed into DH5α Escherichia coli for expression and further analysis. Quantitative GFP analysis showed that pBRAT24C and pBRAT24D induced lower levels of GFP expression than pEAH23A. This suggests that the distance between the lac promoter and gfp does not affect levels of GFP expression and that the sequences found upstream of gfp might affect gene expression through other mechanisms. Further study into the regulatory effects of this region can help maximize BrkA yield from pDO6935, aiding future investigations into its function, structure, and potential as a vaccine candidate.