Developing a system for genetic manipulation of Pelobacter carbinolicus
Pelobacter carbinolicus is a gram negative, non-spore forming bacterial species from the Geobacteraceae family. It is strictly anaerobic and commonly used in microbial experimentation. Its genome has been sequenced and is known to ferment substrates such as ethanolamine, ethanol, and propanediol into acetate and H2 or formate when grown syntrophically with a partner. Despite its wide use, a system for successful genetic transformation in P. carbinolicus has not yet been found. However, other species of bacteria from the same family have been successfully mutated, so it was hypothesized that these protocols could be adapted to develop a system for genetic transformation in our species, which is the focus of this project. Such protocols attempted during experimentation included electroporation, conjugation, and natural transformation. Growth of the bacteria was then attempted in both liquid and plate media sources, and transformation success was determined based on antibiotic control parameters. Competency was not established within my time at the lab, however the best results came from a trial attempted using the conjugation protocol. This trials PCR screening indicated incorporation of the plasmid into the P. carbinolicus genome; however, subsequent sequencing of the mutant strain was inconclusive. Further research into this protocol is required for the establishment of a successful method for genetic transformation within Pelobacter carbinolicus.