Engineering Microbial Communities in Biofilms

Engineering living bacteria within biofilms has remained a challenge owing to difficulty in their cultivation under laboratory conditions due to reliance on syntrophic partners. In order to develop these bacteria for technological use, tools to genetically manipulate them directly, without the need for standard laboratory isolation and cultivation, are required. Here we describe our efforts toward <i>in situ</i> genome editing of autotrophic bacteria in an electrode biofilm. We first demonstrate a CRISPR-based genome editing tool called INTEGRATE in a biofilm-forming marine bacteria, <i>Marinobacter atlanticus </i>in pure culture. Next, we tracked the introduction of a fluorescent marker protein superfolder GFP (sfGFP) into <i>M. atlanticus</i> biofilm cells via conjugation with a fluorescent donor <i>E. coli</i> strain using confocal microscopy. Utilizing a fluorescent <i>M. atlanticus</i> biofilm, the genomic integration of sfGFP in the cells could be monitored in real-time. Lastly, we targeted an electroautotroph <i>Candidatus</i> Tenderia electrophaga that exists in a consortia of ca. 20 other species, and can only grow from CO₂ fixation and electron uptake from an electrode, and showed, for the first time, successful genome integration of a fluorescent marker protein in this uncultivated electroautotroph. This work represents the first step towards <i>in situ</i> genome editing of microbial communities that form electroactive biofilms as an approach towards developing living catalytic materials.