Shewanella oneidensis 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 Escherichia coli, the available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in S. oneidensis 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 (oriT) 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 S. oneidensis MR-1, enabling control over the expression of two different fluorescent proteins. This plasmid toolkit was further used for overexpression of the MtrCAB porin-c-type cytochrome complex in the S. oneidensis ΔmtrA 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 S. oneidensis. 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 S. oneidensis MR-1.

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