Date of Award

12-2017

Document Type

Thesis

Primary Advisor

Autumn Sutherlin

Secondary Advisor

Sarah Lee

Committee Reader

Qiang Xu

Abstract

All organisms are capable of synthesizing isopentenyl pyrophosphate (IPP), a precursor to many different biomolecules such as ubiquinone Q8, cholesterol, and β-carotene. Two pathways, the mevalonate and non-mevalonate pathway, synthesize IPP. Unlike most eubacteria that use the non-mevalonate pathway, low-G+C gram-positive cocci bacteria solely use the mevalonate pathway. This pathway differs from mammals at the rate-limiting step of HMG-CoA reductase and is a potential target for future antibiotics against nosocomial infections caused by Enterococcus faecalis. Unique to enterococci is a fusion protein (encoded by mvaE) made of the first enzyme (acetoacetyl-CoA thiolase) and the third enzyme (3-hydroxy-3-methylglutaryl-CoA reductase) of the pathway. This research focuses on the first three enzymes of the pathway and relies on a series of sub-cloning. First, to isolate thiolase from the fusion protein, sub-cloning mvaC, the gene for thiolase, into pET28 was attempted. Secondly, sub-cloning of the gene for the fusion protein (mvaE) and gene for HMG-CoA synthase (mvaS), which is the second enzyme in the pathway, into pDUET was attempted. Each sub-cloning reaction employed a double restriction digest, gel electrophoresis, DNA purification, ligation of insert and vector, transformation, and mini-preparation of plasmid DNA. Despite repeated attempts and troubleshooting, results were inconclusive. This research has led to the refining of the sub-cloning protocol using the pDUET vector, and future research is needed to understand more about the fusion protein in Enterococcus faecalis.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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