Adding Upstream Sequence and a Downstream Reporter to the Bile Acid Inducible Promoter of <I>CLOSTRIDIUM scindens</I> VPI 12708

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 <i>Clostridium scindens </i>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 <i>E. coli</i>. 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 <i>uidA</i> (ϐ- glucuronidase) reporter was demonstrated. However, no <i>E. coli</i>transformants were demonstrated to possess the <i>baiP</i>-<i>uidA</i> gene fusion. The project strategy is plausible and data regarding the bile acid inducible promoter are greatly needed.

Name thesis:degree_name

Master of Science in Biology

Discipline thesis:degree_discipline

Department of Biology

Year

2009

Author dc:creator

• Mason, Bryan Patrick

Contributors dc:contributor

• Dr. Kinchel Doerner (Director), Dr. Rodney King, Dr. Cheryl Davis

• bacterial chemical modifications
• <i>Clostridium scindens</i> VPI 12708 cloning strategies
• bile acid-inducing gene
• Bacteriology
• Biology
• Cell Biology
• Computational Biology
• Genetics
• Molecular, Genetic, and Biochemical Nutrition
• Systems and Integrative Physiology
• Systems Biology

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/99

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1100