A Synthetic Plasmid Toolkit for Shewanella oneidensis MR-1

<i>Shewanella oneidensis</i> MR-1 is a platform microorganism for understanding extracellular electron transfer (EET) with a fully sequenced and annotated genome. In comparison to other model microorganisms such as <i>Escherichia coli</i>, the available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in <i>S. oneidensis</i> MR-1. Here, we constructed and characterized a plasmid toolkit that contains a set of expression vectors with a combination of promoters, replicons, antibiotic resistance genes, and an RK2 origin of transfer (<i>oriT</i>) cassette, in which each element can be easily changed by fixed restriction enzyme sites. The expression cassette is also compatible with BioBrick synthetic biology standards. Using green fluorescent protein (GFP) as a reporter, we tested and quantified the strength of promoters. The copy number of different replicons was also measured by real-time quantitative PCR. We further transformed two compatible plasmids with different antibiotic resistance genes into the recombinant <i>S. oneidensis</i> MR-1, enabling control over the expression of two different fluorescent proteins. This plasmid toolkit was further used for overexpression of the MtrCAB porin-<i>c</i>-type cytochrome complex in the <i>S. oneidensis</i> Δ<i>mtrA</i> strain. Tungsten trioxide (WO₃) reduction and microbial fuel cell (MFC) assays revealed that the EET efficiency was improved most significantly when MtrCAB was expressed at a moderate level, thus demonstrating the utility of the plasmid toolkit in the EET regulation in <i>S. oneidensis</i>. The plasmid toolkit developed in this study is useful for rapid and convenient fine-tuning of gene expression and enhances the ability to genetically manipulate <i>S. oneidensis</i> MR-1.