(294h) Creation of a Firefly Luciferase Reporter Cassette for Use in PCR-Mediated Gene Deletion and Fusion in Saccharomyces Cerevisiae

When exposed to environmental perturbations, organisms increase production of proteins implicated in survival.  Assessing the transcriptional level of one or more of these responding proteins provides valuable information regarding the cellular surroundings. Reporter systems developed for quantification of promoter response typically rely on enzymatic reactions (β-galatosidase and luciferase) or fluorescent molecules (green fluorescent protein - GFP), however deficits in implementation (such as protein extraction for β-galactosidase and the necessity of expensive high-throughput machinery for GFP) severely limit biosensor applications.    The development of in vivo luciferase reporter systems has led to inexpensive, sensitive and accurate promoter assays without the variability reported between in vitro samplings, but current luciferase reporter systems are largely inflexible to modifications to the promoter of interest.  PCR-mediated gene deletion/complementation techniques that overcome promoter inflexibility have been developed for fluorophores-based reporter systems and show promise for extension to luciferase reporters.  In our study, we report the creation of a novel vector system, which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(−SKL)] capable of one-step, in vivo measurements into a promoter–reporter system via PCR-based gene deletion/complementation. Fluorescent microscopy is utilized to confirm cytosolic localization of the luciferase construct under HUG1 promoter. Further, we demonstrate accurate promoter dose–response through comparisons of luciferase assays to that of a similar HUG1Δ::yEGFP1 promoter–reporter system.  Our data indicate that, alongside a significant decrease in experimental time, the luciferase reporter system is shown to be highly reproducible and correlate well to comparable reporter constructs.  Though we have applied this construct directly to DNA damage biosensing, use extends to any application where promoter response levels are desired