Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model organism. There is a lack of knowledge on how this operon is regulated, so the goal of this project was to create a genetic construct consisting of upstream regulatory elements, a bile acid inducible promoter, and a ϐ- glucuronidase reporter. Cloning strategies utilized PCR to amplify desired DNA fragments and sewing methodology to combine DNA fragments. DNA fragments were ligated into plasmids and transformed into competent E. coli. Transformants were evaluated for the desired reporter gene fusion by blue/white screening, additional PCR, and/or restriction digestion. The bile acid inducible promoter was successfully amplified, and the upstream sequence and uidA (ϐ- glucuronidase) reporter was demonstrated. However, no E. colitransformants were demonstrated to possess the baiP-uidA gene fusion. The project strategy is plausible and data regarding the bile acid inducible promoter are greatly needed.
| Identifer | oai:union.ndltd.org:WKU/oai:digitalcommons.wku.edu:theses-1100 |
| Date | 01 August 2009 |
| Creators | Mason, Bryan Patrick |
| Publisher | TopSCHOLAR® |
| Source Sets | Western Kentucky University Theses |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses & Specialist Projects |
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