Fig. 4: Tuning input/output signaling response by modulating kinase and phosphatase levels.

a Implementation of a covalent modification cycle with kinase (EnvZm2) and phosphatase (EnvZm3t10) variants of EnvZ. The expression level of the output can be tuned as a function of both enzymes, and inputs to each that affect their production rate (u_K and u_P). b Tuning output expression through different dosages of kinase and phosphatase DNA. The heatmap shows the median level of output for each combination of kinase and phosphatase DNA dosages, assayed with poly-transfection⁴⁵ (see Supplementary Figure 14 for full data). The line plots show the same data but are broken out by rows or columns. Brighter lines correspond to bins with increasing phosphatase (left) or kinase (right). c Tuning output expression through small molecule-induced degradation of the phosphatase. DDd is fused to the N-terminus of the phosphatase (see Supplementary Figure 10 for different arrangements and comparison with DDe/4-OHT). The addition of TMP stabilizes the DDd-phosphatase fusion protein⁴⁶. The data are extracted from the full poly-transfection results shown in Supplementary Figures 10 and 11, selecting the middle phosphatase bin (P Marker ≈ 10⁶). The line plots show the same data but broken out by rows or columns. Brighter lines correspond to samples with increasing TMP concentration (left) or bins with increasing kinase (right). All data were measured by flow cytometry at 48 hours post transfection. HEK-293 cells were used for b and HEK-293FT for c. All error bars represent the mean ± s.d. of measurements from three experimental repeats. All heatmap values represent the mean of measurements from three experimental repeats. Source data are provided as a Source Data file.