Chemical tuning of the autonomous timer circuit within Escherichia coli MG1655 AT. (A) Schematic overview of aTc and Van-based tuning of the autonomous timer circuit. (B) Kinetics of the fold change of average cellular YFP fluorescence of MG1655 AT grown on agarose pads under indicated timer tuning conditions and induced at t = 0 h with 100 μM Cuma, relative to similarly grown MG1655 AT cells without induction. The black line is set at 10-fold the basal YFP intensity level of the non-induced control. The data of two independent repetitions was fitted with a Hill function with errors indicated though 95% prediction intervals. The mean of every datapoint is represented by the respective symbols. For each condition, growth of ca. 60 single cells into ca. 100-cell microcolonies was monitored. (C) Delay in YFP expression estimated by the time it takes for the fitted data in panel B to reach 10-fold the basal YFP intensity level of the non-induced control (i.e. black line in panel B). Error bars indicate the variance by which the upper and lower end of the 95% prediction interval cross the threshold.