Rothia mucilaginosa (Rm) is a gram-positive bacterium residing in the oral cavity. Recent studies in our laboratory have shown that this microorganism is able to cleave gluten, including immunogenic domains implicated in celiac disease. This can be beneficial to patients with celiac disease because exploitation of Rm can provide a novel mode of treatment. The enzymes responsible for this cleavage are as yet unknown. The purpose of this study was to optimize the transformation efficiencies of Rm cells through electroporation, with the ultimate goal to create knock-out mutants for enzyme activity. We have determined various aspects of Rm cells relevant for this project: (1) the growth curve characteristics of Rm; (2) the presence of endogenous restriction enzyme activities; and (3) the conditions facilitating Rm electroporation by varying electroporation voltages. Furthermore, electroporation and transformation of the plasmid pUC18 was conducted in Escherichia coli. The growth curve of Rm cells in BHI growth medium incubated at 37°C while shaking showed a doubling time of approximately 3 hours in the logarithmic growth phase. Using a cell sonicate of Rm cells incubated with Lambda DNA and four different restriction enzyme buffers, we found that there were no apparent endogenous restriction enzyme activities detectable. For the electroporation experiments, we used previously published protocols for the bacterium Staphylococcus aureus, as a standard condition to electroporate Rm cells. Those studies have shown that changing electrical parameters during the electroporation would yield a high efficiency rate of gram-positive bacterial transformation (Lofblom et. al., 2006; Metzler et. al., 1992). Therefore in our study, we increased the field strengths (kV*cm-1) to electroporate Rm cells. Rm cells could not be successfully transformed, and we observed that field strengths exceeding 18 kV*cm-1 destroyed Rm cells. On the other hand, the transformation of E. coli with pUC18 was successful. Our studies have laid the groundwork for investigating the transformation of Rm cells, and future studies can use the results obtained to further investigate ways to optimize transformation of Rm cells for potential utility in celiac patients.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/13162 |
| Date | 24 September 2015 |
| Creators | Lee, Ji Youn |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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