Cell-free synthesis and assembly of biomolecular condensates: Engineering properties, functions and regulation

ongoing

Compartmentalization is a defining characteristic of life and has the potential to enable and improve engineered manufacturing routes in biotechnology. Many biomolecules like proteins and RNA have the ability to spontaneously cluster in molecularly dense, phase-separated liquid-like assemblies, termed biomolecular condensates. Biomolecular condensates are promising as synthetic compartments in cell-free reactions and living cells because they could provide programmable, self-assembled spatial organization and rapidly appear or dissolve on demand. However, we are still lacking key engineering and characterization tools, a fundamental understanding of how the unique material properties influence internal biochemistry, and strategies to regulate these dynamic molecular assemblies. I have recently discovered that different condensate-forming proteins can be synthesized and assemble into liquid-like droplets in cell-free transcription and translation reactions run in a custom-designed microfluidic device. This project will pioneer cell-free synthesis for the engineering and characterization of biomolecular condensates, and engineer new synthetic compartmentalization strategies for cell-free systems and living cells. First, developing and taking advantage of a highly controlled microfluidic cell-free environment we will generate and characterize new synthetic compartments with tailored properties. Secondly, we will specifically target molecules and reactions into the condensate phase and systematically study how condensate properties influence biological functions. Finally, we will implement dynamic feedback control mechanisms that can autonomously adjust presence and functions of synthetic compartments in cell-free systems and in cells. SYNSEMBL will break new grounds for applications of biomolecular condensates in material science and synthetic biology.