Date of Graduation

7-2020

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Ivey, D. Mack

Committee Member

Kral, Timothy A.

Second Committee Member

Rojas, Alejandro

Third Committee Member

Lessner, Daniel J.

Keywords

Bioinformatics; Clostridium difficile; Infectious disease; Modeling

Abstract

Clostridioides (formally Clostridium) difficile is a medically relevant pathogen pertinent to infectious disease research. C. difficile is distinctly known for its ability to produce two toxins, enterotoxin A and cytotoxin B, and the propensity to colonize the mammalian gastrointestinal tract. It is known that metabolism is tightly correlated with sporulation in endospore producers such as C. difficile, but an interesting and novel regulatory relationship found by the Ivey lab has yet to be understood. The relationship explored in this study is observed between the sporulation factor, SpoIIE, which represses expression of an ABC peptide transporter, app. In this study, two primary approaches were taken in order to investigate this interaction. The first method involved directly mutating the gene for SpoIIE in attempt to understand which portion of the gene is crucial for retaining protein functionality, in this case, the repression of app. The two genes of interest were readily available on their own individual plasmids, which easily allowed for mutagenesis experimentation before transformation into E. coli. The plasmid with app was constructed to include a fluorescent probe, allowing the relative level of repression to parallel the qualitative measurement of fluorescence. On the SpoIIE containing plasmid, missense point mutations, done using PCR site-directed mutagenesis, and a large deletion of the SpoIIE protein transmembrane anchor, done by endonuclease restriction digest, were performed in pursuit of this investigative method. The secondary approach was to computationally model the SpoIIE protein structures resultant of the genetic mutations done in vitro. Side-by-side images of the predicted mutant models produced from the experimentally preferred algorithm were compared to the wildtype SpoIIE model. The visual analysis of the structures and the comparison of various algorithms is anticipated to be insightful for not only this project, but future exploration into SpoIIE as a multifunctional protein within C. difficile.

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