Supplementary Figure 5: Development and characterization of inter-cellular communication systems based on chemical wires histamine (W_H) and dopamine (W_D).

a) Logic description, schematic symbol and performance of chemical wire-dependent AND-logic gate modules in HEK293T cells. Cells engineered with the I_HRH2 AND W_H-logic-gate module function as W_H-receiver cells secreting the reporter protein SEAP (O_S) in response to W_H. Similarly, the I_DRD1 AND W_D-logic-gate module functions as W_D-receiver cells. Partial data are reproduced from Supplementary Fig. 2a. b) Biosynthetic pathway of W_H production using histidine decarboxylase (HDC) (left). HEK293T cells were engineered with constitutive expression vectors of a truncated version of histidine decarboxylase (tHDC) or mCherry as control and incubated for 48 h. In a next step, the supernatant was transferred to W_H-receiver cells and incubated for another 24 h until SEAP activity was quantified (middle). Different amounts of constitutive tHDC- or mCherry-producing HEK293T cells were cocultured with a fixed W_H-receiver cell amount and incubated for 48 h after SEAP activity was quantified in the supernatant of cells (right). c) Biosynthetic pathway of W_D production using tyrosine hydroxylase (TH) and aromatic amino acid-decarboxylase (AADC). In addition, GTP cyclohydrolase (GCH1) produces the cofactor tetrahydrobiopterin (BH4) required for TH-dependent catalysis (top). HEK293T cells were engineered with constitutive expression vectors of TH-2A-AADC or mCherry as control and incubated for 48 h. In a next step, the supernatant was transferred to W_D-receiver cells and incubated for another 24 h until SEAP activity was quantified (bottom left). Different amounts of constitutive TH-2A-AADC- or mCherry-producing HEK293T cells were cocultured with a fixed W_D-receiver cell amount and incubated for 48 h after SEAP was quantified in the supernatant of cells (bottom right). Error bars represent the means ± s.d. of n=3 independent experiments performed in triplicate.