(563d) Synthetic Biology Framework for Engineering Post-Translational Circuits

Development of signaling circuits that respond to biologically relevant ligands with the same speed and tight control inherent in native signaling pathways would transform our ability to reprogram cellular behavior. Synthetic circuits reported to date have employed regulatory mechanisms such as transcription or translation which are easier to engineer than native signaling pathways but have slower response times and more limited outputs. In a typical native signaling pathway, receptor activation triggers phosphorylation of a protein kinase that in turn phosphorylates many other proteins. This subsequently triggers a wide range of events, including changes to the localization or stability of proteins and the modulation of signaling pathways and gene expression patterns. We have developed novel post-translational synthetic circuits that utilize the same reversible phosphorylation-based mechanism as native signaling pathways, which enables a fast and layered response that facilitates tighter control of cellular behavior. When connected upstream to synthetic receptors, engineered signaling pathways can be configured to sense and respond to changes in extracellular ligand concentration. These circuits are completely orthogonal but can be easily integrated with native signaling pathways. They are also modular and tunable, which enables the same full range of outputs as native signaling pathways. This work introduces a broadly applicable, synthetic biology framework for engineering post-translational regulation, enabling user-defined reprogramming of a cell’s ability to interact with its local environment.