Remarcik, Clint Michael2013-01-302013-01-302010-12https://hdl.handle.net/11299/143710University of Minnesota M.S. thesis. December 2010. Major: Microbial Engineering. Advisor: Dr. Daniel R. Bond PhD. 1 computer file (PDF); vi, 63 pages.The Geobacteraceae are a family of deltaproteobacterial anaerobes known to play important roles in environmental Fe(III)-reduction, subsurface petroleum bioremiediation, bioprecipitation of heavy metals, and reduction of anodes in microbial fuel cells. Electron transfer by Geobacteraceae requires formation of a multicellular biofilm, as cells must attach to surfaces or insoluble particles to bring redox proteins in contact with their electron acceptor, and growth of daughter cells requires cell-cell contact to facilitate longer-range electron transfer. The primary aim of this thesis was to develop new tools for three-dimensional visualization of gene expression within these biofilms. To detect differential expression in a developing biofilm, the genetically tractable representative Geobacter sulfurreducens was utilized as a model. Promoters from two c-type cytochromes proven to be important in electron transfer were inserted upstream of the fluorescent protein mCherry, or a derivative of mCherry containing an additional peptide sequence (LVA) to accelerate protein degradation and act as a signal of more recent expression. Control plasmids containing no mCherry, or with the fluorescent protein under control of a constitutive taclac promoter were also transformed into G. sulfurreducens. The plasmid backbone used for expression was found to affect growth of cells as biofilms, and a new mobilizable plasmid was developed for use in these experiments. Cells had to be exposed to oxygen for four hours to achieve maximum fluorescence, indicating that future real-time studies with these proteins reliant on oxygen leakage into biofilms would not be reliable. In addition, as G. sulfurreducens was found to possess significant background fluorescence when grown as a biofilm in the same emission range as mCherry, future studies should investigate reporters that operate at different wavelengths. After correcting for these effects, it was demonstrated the promoter for omcZ was expressed fairly uniformly throughout the biofilm, with localized pockets of high expression. The promoter for omcS, was expressed at significantly higher levels towards the top of the biofilm when biofilms were at maximum thickness (>20 μm). These results demonstrate that promoter-reporter fusions can be used for visualization of cytochrome expression in G. sulfurreducens biofilms, and highlights numerous issues related to plasmid choice, protein oxidation, and background fluorescence which could create artifacts and confound results.en-USMicrobial EngineeringThesis or Dissertation