Investigating the Impacts of Mutations On Promoter Efficiency

Abstract

Promoters are specific sequences of nucleotides that serve as initial determinants for the intensity of gene expression. Promoter variation has been shown to contribute to development of genetic disorders and cancer. Our objective was to investigate the impact of promoter variations on bacterial gene expression. We hypothesized that nucleotide changes would impact expression and regulation of reporter genes. To test this hypothesis, we used recombinant DNA technology to combine wild-type and mutant versions of the pLacI promoter with a characterization construct encoding green fluorescence protein (GFP) followed by the LacI repressor under the control of an arabinose inducible promoter in a plasmid. The resulting constructs were transformed into Escherichia coli. The wild-type promoter acted as our control while two mutant promoters each contained two or three nucleotide changes. To determine the efficiency of each promoter, we quantified GFP expression by measuring fluorescence. Both mutants exhibited significantly reduced fluorescence, demonstrating small nucleotide changes significantly impact gene expression. To determine if mutations impact repression, different concentrations of arabinose were added to growing cultures. Reduced fluorescence was observed in all samples, suggesting the mutations did not impact repression. Collectively, our data demonstrates that nucleotide changes in promoters can drastically impact gene regulation.