Redesigning gfp Reporter System for Utilization in Clostridium Difficile

Clostridium difficile (C. difficile) is a gram-positive bacterium that comprises part of the healthy human gut microbiome. When it gains sufficient access to peptides, C. difficile flourishes and releases tissue-damaging toxins, which cause inflammation of the colon that can develop into a Clostridium difficile Infection (CDI).10 The Ivey Laboratory believes that the best tactic in preventing CDIs is stopping peptide ingestion, which theoretically could be accomplished by manipulating the oligopeptide permease (App) system.7 In order to verify that altering the App system would successfully impede peptide uptake, first the expression of the app Promoter Region (appProR) of C. difficile’s DNA needs to be better understood. This characterization can be accomplished by fusing appProR to the gfp-reporter gene, which codes for Green Fluorescent Protein (GFP). GFP emits green fluorescent light when exposed to blue or ultraviolet light, and the degree of fluorescence can be used to quantify the gene expression of whatever DNA sequence to which the gfp-reporter gene is fused.9The specific aim of this project was to incorporate the appProR-gfp-reporter gene complex first into Eschericheria coli (E. coli), and then into Bacillus subtilis (B. subtilis). Those two bacterial species were chosen as hosts for the transformations, for E. coli and B. subtilis are known for being more receptive to recombinant DNA techniques than C. difficile.22 By ligating the appProR-gfp-reporter gene sequence of pUA321 to pG+host4, the resulting plasmid, pUA625, contained a broad enough host range to transform both gram-negative E. coli and gram-positive B. subtilis. Those successful transformations indicate that pUA625 could be integrated into C. difficile in the future, an achievement which would lead to a better understanding of the expression of C. difficile’s App system.