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Cloning the omcF Gene from Geobacter sulfurreducens to E. coli: A Path to Efficient Bioreactors

This project aims to clone the omcF gene, aiding electricity production in Geobacter sulfurreducens through microbial nanowires. The procedure involves DNA extraction, PCR amplification, and transformation into E. coli for potential bioreactor use.

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Cloning the omcF Gene from Geobacter sulfurreducens to E. coli: A Path to Efficient Bioreactors

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  1. Cloning the omcF gene from geobactersulferreducens to E. coli Valerie Wisco & Casey Durnan

  2. Geobactersulferreducens • Anaerobic bacteria • Has the ability to transfer electrons outside the cell and transport the electrons over long distances by conductive filaments known as microbial ‘nanowires’ • have the ability to transfer electrons on to the surface of electrodes creating a pass of electricity • Has the highest known current density of any pure culture

  3. We are receiving our cultures from a research lab from the University of Masachusetts – Lab Manager Joy Ward • Will be sent in and cultured anaerobically in NBAF (acetate fumarate) media

  4. omcF • Accession #:AAR35805.1 • 315 base pairs, no introns • The omcF gene helps code for a subunit of a large lipoprotein cytochrome-c. It is the heme-binding site. • Shown to help regulate the transcription of other Omc genes that play a role in current production • Trials indicate down-regulation of omcF profoundly inhibits electron transfer, reducing electricity production in the organism. • Cloning this gene can eventually help produce efficient bioreactors.

  5. Tentative Procedure • Grow source bacteria culture on NBAF media • DNA extraction • PCR amplification • Quality check/ purify DNA by electophoresis • Digestion of source DNA and promoter • Ligation • Digestion/Ligation of new part and plasmid • Transformation into E. Coli host • Antibiotic selection-Kanomycin • Possible verification testing

  6. Internal Restriction

  7. Primer Design

  8. Primer Design

  9. Primer Design • Verified removal of restriction site • Verified lack of new restriction sites • Verified protein sequence

  10. Vector and Promoter Selection • Promoter: pBad/araC • Strictly controlled by L-arabinose as an inducer and restricted by araC • Vector: • PSB2K3 • KanomycinResistent

  11. Testing • SDS-PAGE • Eventual testing would be to see if electrons would be passed onto an electrode, creating a current- after cloning of all essential parts, all Omc genes and genes for creating the “nanowires” • Other genes that could be cloned would be OmcZ (essential for current production), OmcS (conducts the electrons away from the cell-on the pili) and OmcS (important in electron conduction to anodes)

  12. References • Kim, B., Postier, B., DiDonato, R., Chaudhuri, S., Nevin, K., & Loveless, D. (2008). Insights into genes involved in electricity generation in geobactersulfurreducens via whole genome microarray analysis of the omcf-de!cient mutant. Bioelectrochemistry, Retrieved from http://www.geobacter.org/publication-files/18538641.pdf • http://www.ncbi.nlm.nih.gov/nuccore/NC_002939.5?report=genbank&from=2667181&to=2667495&strand=true---- • http://www.geobacter.org/about

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