Gene transfer plays an important role in bacterial evolution. Especially in an under explored species like Rhodococcus, a type of bacteria found in the soil. Rhodococcus has several applications in the pharmaceutical industry and in the production of antibiotics. Rhodococcus possess several unique sets of properties which makes it beneficial to have a reliable method of producing mutants of Rhodococcus. The goal of the experiment was to find an efficient way of forming Rhodococcus colonies with kanamycin resistant genes. The project began from an unexpected observation from an earlier experiment with Rhodococcus strain MTM3W5.2. where I attempted to transform this strain with a transposon via electro-transformation. The colonies that grew/ appeared transformants were screened to confirm the presence of kanamycin gene, however there was no amplified DNA seen on the PCR gel (i.e. absence of the kanamycin gene). The electro-transformant colonies were selected on LB plates containing different higher concentrations of kanamycin. Then the appeared transformants were again screened via disk diffusion assay and were classified into 3 different kanamycin resistant phenotypes. Majority of the “C” phenotypic colonies (i.e., high level resistance to kanamycin) appear to contain the kanamycin gene, but these colonies were less in numbers. This led us to try another method of gene transfer which is conjugation. Conjugation was carried on a double selection antibiotic plate containing both chloramphenicol (30 µg) and kanamycin (100 µg). The transconjugate colonies that appeared on the double selection plates were also screened by PCR, but none of the colonies had amplified DNA suggesting absence of the kanamycin gene. The colonies seen on the double selection plate were possibly due to spontaneous mutation or some type of unknown phenotypic variation. However, in the future, double selection plates with higher concentrations of antibiotics can possibly give us transconjugants with kanamycin genes.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:honors-1692 |
| Date | 01 May 2020 |
| Creators | Kapadia, Jaimin Maheshbhai |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Undergraduate Honors Theses |
| Rights | Copyright by the authors., http://creativecommons.org/licenses/by-nc-nd/3.0/ |
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