Staphylococcus aureus is the most prevalent pathogenic bacterium for causing serious infectious diseases in the United States with over 600,000 cases reported annually. Superantigens (SAgs) are an important virulence factor secreted by most S. aureus strains. SAgs encoded in the enterotoxin gene cluster (egc) of S. aureus strains are frequent and essential for the development of infective endocarditis, a serious and potentially fatal bacterium infection in the heart endothelial lining and heart valves that causes significant vegetations as found in vivo rabbit models. Infective Endocarditis occurs in 30-60% of cases with S. aureus infection with a reported mortality of 20-40%. Regulation and inducible conditions for the egc-encoded superantigens are still not fully understood and are hypothesized to be produced locally to promote disease by establishing the site of infection. Previous studies have shown egc superantigens exhibiting low expression occurring in early exponential phase in vitro.  Master transcriptional regulators of S. aureus virulence factors were selected for overexpression with a two-plasmid vector system in S. aureus MN8, a methicillin susceptible strain. Constructs with a two-plasmid vector system contain a primary plasmid encoding a superantigen promoter with a fluorescent reporter and a secondary plasmid encoding a xylose-inducible promoter-repressor with a select master regulator for overexpression. Constructs will be tested in vitro through batch testing and single-cell analysis to examine regulation and required inducible conditions of egc-encoded superantigens. We hope this study will provide insight on egc superantigens expression that contribute to serious S. aureus infections and the development of infective endocarditis.