Figure 4.

Real-time control of the combined CRISPR and antisense RNA system. Repression of GFP was first mediated by activating the CRISPR system only in cells (containing sgR14-T6 and asRS6; see Figure 1C for the schematic figure) with Abs = 0.05 at t = 0 h (0.2 ng/ml aTc and 0.25 mM IPTG; both filled black and gray circles). A significant difference in repression was observed by 3 h, determined using the independent sample t-test (t = 10.25, P < 0.01) (47). At t = 7 h (top), 9 h (middle) and 11 h (bottom), cells were transferred into fresh media (diluted back to Abs = 0.05) that maintained both CRISPR and asRNA activation (0.2 ng/ml aTc, 0.25 mM IPTG and 5 mM Ara; filled black circles). A significant difference in derepression was observed by 3 h after the transfer, based on the independent sample t-test (t = 11.76, P < 0.01) (47). Derepression efficiencies (92%) and the times by which the system responded (3 h) were independent of the duration of the repression. GFP is under the control of the constitutive Bba J23104 promoter. Fluorescence (a.u.) was measured using flow cytometry. The error bars represent the standard deviation of the fluorescence values from three biological replicates performed on three different days. Open circles represent cultures with no inducer.