Figure 2.
Constructing gene circuits using cooperative TF assemblies enables expanded steady-state signal processing behavior. (A) Computational model-driven approach for exploring engineerable circuit behavior. Our parts collection (left) defines available configuration space (middle: PDZ-ligand variants, Kp; synTF-DBM variants, Kt; clamp/synTF/DBM units, nc). Our model (fig. S7–9) computes input-output functions for this space, mapping the potential circuit behavior range (behavior space, right). (B) Programmed complex assembly enables tuning of single-input circuit dose response. For a single-input (2-node) circuit, synTF is induced by ATc addition (input node) and assembles with constitutively-expressed clamp to regulate GFP transcription (reporter node, left). In model-computed circuit behavior space (right) colors indicate different complex sizes (nc). Five circuits with different assemblies (parameters: Kt affinity in blue, Kp affinity in red, nc), were constructed and tested by inducing with ATc and measuring GFP by FACS after 16 h (below). Points represent mean values for three experiments ± SE. (C) Programmed complex assembly enables tuning of two-input dose response between linear and non-linear computations. In a two-input circuit, synTFI and synTF2 are induced by ATc and EST respectively (input nodes), assembling with constitutively-expressed clamp to regulate a downstream reporter node (left). Behavior space for the full set of available circuit configurations (center) are plotted as Kullback-Leibler divergence (DKL): “similarity” between model-computed output surfaces and archetypal Boolean AND and OR surfaces (see fig. S13). Grey areas in the plot indicate regions of AND- and OR-like behavior. Selected circuits, with corresponding reporter complex parameters (Kt affinity, blue; Kp affinity, red; nc), were constructed and their 2D output surfaces experimentally measured (right) by inducing with ATc and EST and measuring GFP by FACS after 16 h (see Supplementary Materials and Methods).