Programming Synthetic Feedback Using Designer Proteins

Feedback plays a key role in nearly all biological processes, from the cell cycle to chemotaxis. Despite the clear importance of feedback to cells, synthetic biologists have yet to invent a truly modular device for performing feedback on proteins. Existing methods for performing feedback have been designed for control of specific pathways or molecules, with limited tunability. To this end, we leveraged recent advancements in protein design to design a completely de novo, static, five-helix “Cage” with a single interface that can interact either intra-molecularly with a terminal “Latch” helix or inter-molecularly with a peptide “Key”. Encoded on the Latch are functional motifs for binding, degradation, or nuclear export that function only when the Key displaces the Latch from the Cage. Using one of these designer switches, degronLOCKR, we were able to regulate degradation of a variety of cargoes, including transcription factors, dCas9, and kinases. The modularity afforded by the de novo designed LOCKR switches offers distinct advantages over previous efforts to engineer cellular circuits, which have been limited to repurposing modular protein domains from nature. Leveraging the plug-and play nature of degronLOCKR, we implemented feedback control on both endogenous signaling pathways and synthetic gene circuits. We first generated synthetic negative feedback in the yeast mating pathway via fusion of degronLOCKR to endogenous signaling molecules, illustrating the simplicity with which this strategy can be used to rewire complex endogenous pathways. We next benchmarked degronLOCKR-mediated feedback control on a synthetic gene circuit to quantify its feedback capabilities and operational range. The designer nature of degronLOCKR enables simple and rational modifications to tune feedback behavior in both the synthetic circuit and the mating pathway. De novo protein design promises to greatly expand the realm of possibility of synthetic biology with a toolkit of composable, modular, and tunable parts that are also bio-orthogonal