Engineering Flourescent Fusion Protein with Novel Electron Uptake Gene in Shewanella oneidensis

Abstract

By Anna Cunnane, Biological Sciences; Josh Sackett, University of Cincinnati

Advisor: Annette Rowe

Presentation ID: 145

Abstract: Mineral reduction via extracellular electron deposition onto solid phase minerals and/or electrodes has been well characterized in several organisms, but the reverse of this process has yet to be characterized. For example, the MTR respiratory pathway in the Shewanella oneidensis strain MR-1 is known to be involved in electron deposition, but only accounts for some of the electron uptake under aerobic cathodic conditions. Using S. oneidensis as a model system, our work recently identified five genes that are uniquely involved in electron uptake and not electron deposition, however the functional role of these proteins is unclear. One of these genes, SO3662, is thought to produce a ferredoxin (Fe-S protein) involved in extracellular electron uptake. We predict SO3662 associates at the cytoplasmic membrane and helps transfer electrons to the terminal cytochrome oxidase to allow for energy conservation. This research aims to characterize the localization of this protein by generating gene fusion with the fluorescent marker cyan fluorescent protein (CFP). SO3662 was PCR amplified and inserted into a custom E. coli plasmid that contained CFP. The plasmid was cut on the N-terminal end of the CFP and the gene was inserted in-frame at restriction enzyme sites. The plasmid was transferred from E. coli into S. oneidensis via conjugation. SO3662 will be localized in the cell using live fluorescent microscopy techniques, and possibly TEM. Results of this work will help inform the role SO3662 plays in electron uptake and could help better engineer or optimize this system for microbe-electrode technologies such as electrosynthesis.