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

Introduction

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.

Significance of 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
• Rapid appearance or dissolution 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.

Recent Discoveries

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.

Project Objectives

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. The main objectives include:

1. Developing and taking advantage of a highly controlled microfluidic cell-free environment to generate and characterize new synthetic compartments with tailored properties.
2. Specifically targeting molecules and reactions into the condensate phase and systematically studying how condensate properties influence biological functions.
3. Implementing dynamic feedback control mechanisms that can autonomously adjust the presence and functions of synthetic compartments in cell-free systems and in cells.

Expected Impact

SYNSEMBL will break new ground for applications of biomolecular condensates in material science and synthetic biology.