Synthetic biology approach towards engineering of Shewanella oneidensis MR-1 for microbial fuel cell technologies

In the past decade the emerging field of microbial electrochemical technologies (METs) has gained increased attention due to its potential for bioenergy production and bioremediation. By utilizing pollutants or waste as carbon sources electroactive bacteria (EAB) can convert chemical energy into electricity, thereby conceivably closing the waste disposal energy generation loop. These EABs can generate current anaerobically by forming an electroactive biofilm on conductive electrode materials via extracellular electron transfer (EET). The genetically tractable EAB model organism Shewanella oneidensis MR-1 (SOMR-1) already possesses several EET routes and a large respiration versatility. These traits make it feasible as a synthetic biology chassis to increase predictability, stability and novel functionalities of MET applications. However, as synthetic gene circuits become more elaborate in size and complexity and only relatively few well-characterized biological parts have been described for this organism, precise genetic engineering increasingly presents a bottleneck for this new technology. Here, the synthetic biology toolbox for SOMR-1 was expanded by establishing the Standardised European Vector Architecture (SEVA) plasmid platform providing characterisation of plasmid maintenance with a large range of replication origins, quantification of plasmid copy numbers and their compatibility as multi-plasmid bearing systems in SOMR-1. Further, establishment of transcriptional regulation using oxygen independent inducible promoters was realised. In this work the novel cyclohexanone inducible promoter PChnB/ChnR was introduced among others and characterised using oxygen independent reporter assays. A synthetic flavin gene operon under the control of PChnB/ChnR was used to show enhancement of SOMR-1 EET in small-scale MFCs using screen-printed electrode technology. Additional screening methods are presented which were aimed to identify novel EET capabilities in SOMR-1 using a colorimetric tungsten trioxide (WO3) assay