P167: Variable expression plasmids for controlling protein production in the electrogenic bacteria Shewanella oneidensis and Geobacter sulfurreducens

Bacteria that oxidize or reduce metals fundamentally shape the environment by performing crucial roles in metal cycling. Two dissimilatory metal-reducing genera, Shewanella and Geobacter, are studied because they are able to respire insoluble surfaces including Fe(III) oxides and electrodes. This transfer of electrons from intracellular metabolism to the extracellular environment provides the potential for bioprecipitation by altering the solubility of heavy metals or for biosensor applications by utilizing electron flow to an electrode in response to a signal. Genetic systems for the manipulation of Shewanella and Geobacter exist but there is limited experimental evidence to allow predictable and tunable control of gene expression. In this project, modular promoter and ribosomal binding site (RBS) combinations were created to produce a suite of inducible expression plasmids leading to low, medium and high levels of protein production. Shewanella oneidensis MR-1, Geobacter sulfurreducens PCA and Escherichia coli UQ950 were transformed with these plasmids. Protein production was evaluated by enzymatic assay and correlation of promoter and RBS strength demonstrated between the three species. These plasmids were then used to increase flavin synthesis and export in S. oneidensis. Shewanella species can reduce a variety of extracellular substrates efficiently by secreting flavin-based electron shuttles. Increasing flavin production and secretion accelerated respiration of insoluble electron acceptors. The ability to predict and control protein production will facilitate laboratory study of these microbes and increase their utility in future biotechnology applications.