Fig. 3: Tunable eCRISPRa within an ‘artificial biofilm’.

a Schematic illustration of the electrogenetic CRISPRa system powered by the oxyRS regulon. Electroinduced gRNA sg108 (from plasmid pSC-O108) forms a complex with constitutively-expressed dCas9ω to initiate transcription of the gene-of-interest (GOI) in plasmids pMC-GFPmut2 or pMC-LasI-LAA. b General workflow and setup for experiments with ‘artificial biofilms’. After electrodeposition, we thoroughly washed the film with PBS to remove excess cell/PEG-SH solution. 150 μL of 20% LB was then added into the well to submerge the film in growth media. c Confocal images of E. coli harboring the eCRISPRa-GFP genetic cassette that were embedded in the PEG-SH film. Varying times of electroinduction and the resulting charges (millicoulomb; mC) applied to the cells are indicated below images. Neg Ctrl: negative control. 200 μM of H₂O₂ was exogenously added to culture media to activate the oxyS promoter as a positive control (Pos Ctrl). Top: brightfield; Middle: Merged; Bottom: FITC filter. Scale bar = 50 μm. d Percentage of E. coli in the PEG-SH film that were activated through eCRISPRa. Data are quantified from images in Fig. 3c with ImageJ and presented as mean ± s.d. (n = 4). Comparisons used ordinary one-way ANOVA. e AI-1 assay indicating the amounts of AI-1 generated via CRISPRa lasI cells. Brown: 0 min electroinduction. Dark green: 15 min electroinduction. Light green: 30 min electroinduction. Data are presented as mean ± s.d. (n = 4). Comparisons used ordinary one-way ANOVA. f Measured fluorescence in a coculture comprised of eCRISPRa lasI cells (in film) and AI-1 fluorescent reporters (NEB10β + LasR_S129T-GFPmut3) situated in the liquid above. Data are presented as mean (n = 2). Comparisons used unpaired, two-sided t-test. In (d)–(f), P values were calculated between electroinduced samples and the uninduced control. Exact P values are provided in Source data. Individual replicates were indicated as open circles.