Fig. 1. Design of composable protein circuit components.

(A) Composable protein units (partial circles, left) can regulate one another in arbitrary configurations with diverse functions (middle). Protein-level circuits can interface directly with endogenous protein pathways and operate without modifying the genome or entering the nucleus (right). (B) (Left) The protease-activatable reporter (green) is stabilized by removal of a DHFR degron (black target) through protease (partial circle) cleavage of a corresponding target site (yellow circle).TMP (blue diamond) inhibits the degron and thus stabilizes the reporter. (Middle) Flow cytometry distributions of reporter fluorescence with (purple) or without (orange) TEVP. Distributions are limited to the gated area in fig. S1A. Solid curves indicate skew Gaussian fits. Vertical dashed lines and stars indicate distribution modes, which are plotted in subsequent figure panels. (Right) Analysis of reporter response to TMP and/or TEVP. Each dot represents one replicate. Stars indicate data from the middle panel. a.u., arbitrary units. (C) In the protease-repressible reporter, protease cleavage exposes an N-end degron (covered target) to destabilize the reporter. (D) Three proteases (columns) exhibit orthogonal regulation of three reporters (rows). Mean fluorescence intensity of three independent measurements is normalized to the TMP-stabilized value of the corresponding reporter. (E) Design for protease-repressible proteases. TEVP is split as indicated and then reconstituted through dimerizing leucine zippers (light and dark blue zig-zags). A leucine zipper–tagged HCVP (red partial circle) can dock with the target TEVP and cleave it to remove leucine zippers, effectively repressing TEVP.TVMVP (purple partial circle) can be regulated using the same design. (F) A single-chain variant of the HCV-repressible TEVP allows docking of and repressive cleavage by HCVP. (G) Protease regulation can propagate through a three-stage cascade. Repressible HCVP uses a variant design, in which TEVP cleavage separates core HCVP from its docking leucine zipper and activity-enhancing copeptide (small pie slice). In all panels, red lines indicate triplicate mean.