Synthetic Biomolecular Condensates: Design Principles and Applications.

Synthetic biomolecular condensates are engineered membraneless compartments formed through phase separation of biomacromolecules. Beyond lock-and-key interaction-dependent control of cellular processes, phase separation enables the spatiotemporal organization of defined sets of biomolecules and assembles functional units into mesoscale structures that can reversibly form or dissolve in response to environmental cues. This chapter focuses on the design and engineering of synthetic intrinsically disordered proteins (synIDPs) to build programmable condensation systems for cellular controls. We outline the molecular principles that link IDP sequence features to phase behaviors. The chapter then presents a practical workflow for the development of synIDP-based condensates for cellular controls, including rational design, gene synthesis, protein expression, purification, and biophysical characterization. Finally, we describe synthetic gene circuit-based methods for applying synthetic condensates in E. coli to regulate transcription and translation. Together, this new method demonstrates how engineering synthetic condensates offers a new layer of cellular control for synthetic biology.