Fig. 7. Network integration to enable remote feedback control of eCRISPR activity within the ‘Internet of Life’.

a Automated feedback control of eCRISPRa-regulated gene expression (i) Fluorescence level of the ‘artificial biofilm’ containing engineered eCRISPRa bacteria (NB101 harboring pSC-O108, pdCas9ω, and pMC-GFP; analogous to Fig. 6). Fluorescence measurements were taken every 15 min (0.25 h). Brown (open squares and line): negative control 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 during each yellow zone. The red zone indicates when the selected individual sample was being photobleached. N = 2 individual replicates are shown in open circles and open squares. The green filled circle represents the unbleached control after termination. 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 and proceeded to trigger the remote ‘actuation checkpoint’. b Current threshold (pink dotted line) was set at 1.1 μΑ. The red line indicates when the current threshold was met, which prompted the PC at the remote location to send a text message to users. Filled circles represent the mean of individual replicates (n = 2).