Date of Award
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.
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Harris, Madison Anne, "Sub-cloning Genes of the Mevalonate Pathway from Enterococcus faecalis into pDUET and pET28" (2017). Honors College. 31.