Steps Towards Identifying the Transcription Start Site Upstream of the brkA Gene in pDO6935 Plasmid Using the ARF-TSS Method

Résumé

Type Va autotransporters are expressed on Gram-negative bacteria, where several of these model systems are believed to contribute to virulence. The primary virulence determinant of Bordetella pertussis is accounted for by the autotransporter, BrkA, which facilitates host interactions and displays resistance to the effects of serum. While the brkA gene expressed in the Escherichia coli pDO6935 plasmid has been used in studies addressing brkA's functionality and characteristics, the promoter sequence responsible for brkA’s constitutive expression in E. coli remains unknown. Furthermore, elements of the Lac operon, which have been studied for their role in regulation of gene expression, are positioned upstream of the brkA gene within the pDO6935 plasmid and thus, may serve as the putative promoter in an E. coli (DH5-α) model system. Our study aims to validate procedures established in the adaptor- and radioactivity-free identification of the transcription start site (ARF-TSS) method to map the TSS of the brkA gene within the pDO6935 plasmid sequence. The ARF-TSS method depends on cDNA generation from target gene mRNA. The determining factor depends on reverse transcriptase used during cDNA generation, as well as temperatures used to prevent internal structure formation. To build upon previous work and attempt new temperature bounds, we aimed to introduce the SuperScript^TM IV Reverse Transcriptase during cDNA generation. While unable to replicate the results of a previous study, our findings support the established usage of enzymatic lysis to generate higher RNA yields during RNA extraction, as well as indicate optimal annealing temperatures may lie within the 69-73°C temperature range for PCR following cDNA generation using the SuperScript^TM IV Reverse Transcriptase. The acquired knowledge is valuable for advancing future investigations focused on identifying the promoter of the BrkA autotransporter in an E. coli system expressing pDO6935 using the ARF-TSS method as a reference point.