Figure 4. Evaluation of the functionality of the digitalizer module.

1. Schematic representation of the relevant features of the specific regulatory device used to evaluate the efficiency of the digitalizer module by means of a reporter msf•GFP gene.
2. GFP was used as fluorescence reporter to analyse the OFF and the ON expression profiles of the digitalized version of XylS/Pm system in flow cytometry experiments. The plot represents the median and the SD values of fluorescence from three independent experiments without inducer (t = 0) and upon 3MBz induction along time, at the indicated time points.
3. Representative experiment showing the distribution of fluorescence in cell population under non‐inducing conditions (t = 0) and after induction at the indicated time points (t = 5 min to t = 160 min), as indicated. The region considered as negative for the fluorescence signal is marked between red dashed lines, as assessed by control cells carrying a plasmid with a promoterless GFP (grey plot).
4. Analysis of the population with respect to cell‐to‐cell heterogeneity by means of the coefficient of variation (percentage CV*100) at the off state (t = 0) and along the induction of the system, as described. Data correspond to the mean and SD values obtained from three separate experiments.
5. The NIa protease was used as a sensitive reporter to test both the basal and the induced activity driven by the digitalized XylS/Pm device. The Western blot shows the cleavage of a NIa‐sensitive TpiA target protein under the indicated conditions, which are equivalent as those used for the experiment presented in Fig 2. The control lane #1 was pasted from a different gel to show the location of the intact protein (see a detailed cleavage kinetics in Appendix Fig S6).