Fig. 3

Assessment of SWT orthogonality and a three-layer cascade circuit. (A) Orthogonality characterization of S15 and S16. Each element of the matrix represents the ON/OFF ratio for the indicated SWT/trigger combination. Fold change value was represented by a color scale. (B) Schematic of a three-layer cascade circuit. The input layer X generates T15, which in turn activates the signal processing layer Y (S15-T16) to express T16, enabling the expression of reporter from the final reporter layer N (S16-3WJdB). (C) Characterization of the three-layer cascade circuit. The concentration of the report module N (S16-3WJdB) was fixed at 10 nM, and the concentrations for input X and signal processing module Y were adjusted. (D) Combinatorial test of different components for three-layer cascade circuit. (E) Time-course measurement for the three-layer cascade circuit. For 3D and 3E, X and Y modules were fixed at 20 nM, and N module was fixed at 10 nM. All data shown are n = 3 independent biological replicates. Error bars represent the standard deviation (s.d.) of three biological replicates