Fig. 6: Automated dynamic control of gene expression.

a Schematic illustration of the BioSpark system allowing electrochemical induction and real-time fluorescence/electrochemical measurements. b Automation experiment workflow. ‘Artificial biofilms’ containing engineered bacteria are assembled onto the optoelectrochemical device. The gene expression level, represented by the emitted fluorescence, is constantly monitored by BioSpark and sent to the PC (diamond) for processing. Our custom algorithm (Supplementary Fig. 13d) then determines the status of expression via calculating the rate of fluorescence increase (“Methods”). When expression begins to tail, a user-specified threshold is met, and the potentiostat triggers an induction voltage for a specified charge. The experiment is automatically terminated when the fluorescence level is above a user-defined GFP threshold. c Automated dynamic control of eCRISPRa-regulated gene expression. (i) Fluorescence level within the ‘artificial biofilm’ containing eCRISPRa bacteria (NB101 harboring pSC-O108, pdCas9ω, and pMC-GFP). Fluorescence measurements were taken every 15 min (0.25 h). Experiment was terminated automatically when the mean fluorescence was above the specified target (blue dotted line). Brown (open squares and line): negative controls to which no induction voltage was applied. Yellow zones indicate the durations when the induction voltage (−0.8 V) was applied to the experimental samples, and 0.4 ft³ h⁻¹ of oxygen was supplemented to both the experimental and negative control samples. A total charge of 2 mC was applied in each yellow zone. N = 2 individual replicates are shown in open circles and open squares. For each biological replicate, three fluorescence measurements were performed and averaged. Lines (with no symbols) indicate the mean of biological replicates. (ii) Ratio of slope, S, to S_max computed by our custom algorithm. Ratio threshold (purple dotted line) was set at 0.4. Orange line: algorithm applied the initial induction voltage. Yellow lines: ratio threshold met thus another electroinduction was triggered. Teal dotted line: both the ratio threshold and the induction level target were met; hence no voltage was applied, and the experiment was terminated. Filled circles represent the mean of individual replicates (n = 2).