Rhodococcus, a species of bacteria commonly found in the soil, is a under-explored producer of small bioactive compounds including siderophores, pigments and antibiotics.. MTM3W5.2 is a strain of Rhodococcus that was previously discovered to produce an antibiotic-like compound that has inhibitory effects on other Rhodococcus strains, including the veterinary pathogen, R. equi. The biosynthetic gene cluster responsible for production of the antibiotic has been identified, and a small gene, BTZ20_3964 at the start of the operon is believed to be the regulator of the gene cluster. To test this, a deletion construct was created using an overlap-extension PCR method to remove most of gene 3964. The deletion construct was cloned into the plasmid pEX18Km, along with a kanamycin resistance marker. The deletion construct, pEX18Km3964AD was transformed into competent MTM3W5.2 cells for a double crossover recombination event to replace the functional gene with the deletion construct, using the KanR gene to select for the merodiploids, and the SacB gene to select for the deletion mutants after the second recombination event. The deletion mutant’s ability to produce the inhibitory compound will be determined. Production of the compound will be restored by complementing the deletion mutant with the functional gene cloned into the expression plasmid pDD57.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-1329 |
| Date | 12 April 2019 |
| Creators | Sellick, Katelyn, Lampson, Bert |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | Appalachian Student Research Forum |
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