Steps toward a luciferase assay system for investigating gene expression

The luciferase reporter assay is a powerful method of studying gene expression at the transcriptional level because of its high sensitivity and convenience. It detects the activity of luciferase, a light-producing enzyme encoded by the luxCDABE operon. By cloning promoters of interest upstream of the luxCDABE operon in the pCS26 vector, light production can be correlated to promoter activity. In this study, we developed steps toward a luciferase assay system that can be used to investigate gene expression in a variety of biological models by attempting to establish positive and negative controls within the context of the previously proposed AcrS repression of the acrAB and acrEF operons. To assess this repression, luciferase activity can be compared in the Escherichia coli wild-type BW25113 and ΔacrS JW3232-1 strains transformed with pCS26 vectors fused with promoters. Attempts at cloning the promoters of acrAB and acrEF into pCS26 through Gibson cloning failed, likely as a result of inefficient enzyme activity. We assessed the suitability of ydcWp-pCS26 as a positive control, which contains the promoter of ydcW, an aldehyde dehydrogenase gene, fused upstream of the luxCDABE operon. Through luciferase assay measurements, we determined that ydcWp-pCS26 acts as an appropriate positive control for studying AcrS repression which produces light at consistent levels regardless of the presence of AcrS. ydcWp-pCS26 can be similarly evaluated in future experiments as a potential positive control due to its constitutive expression. We also attempted to create a negative control containing a non-promoter insert upstream of the luxCDABE operon in pCS26 which should not produce light in any condition. Cloning the negative control by
inverse PCR was unsuccessful but should be continued along with construction of the acrABp- and acrEFp-pCS26 constructs in future experiments to create a complete luciferase assay system functional for investigating regulation of acrAB and acrEF and various other biological systems.