Cell-Free Expressed Membraneless Organelles Inhibit Translation in Synthetic Cells

Compartments within living cells create specialized microenvironments, allowing multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation such as P-granules and nucleoli. Synthetic minimal cells are widely used to study many natural processes, including organelle formation. In this work, synthetic cells expressing artificial membrane-less organelles that inhibit translation are described. RGG-GFP-RGG, a phase-separating protein derived from Caenorhabditis elegans P-granules, is expressed by cell-free transcription and translation, forming artificial membraneless organelles that can sequester RNA and reduce protein expression in synthetic cells. The introduction of artificial membrane-less organelles creates complex microenvironments within the synthetic cell cytoplasm and functions as a tool to inhibit protein expression in synthetic cells. The engineering of compartments within synthetic cells furthers the understanding of the evolution and function of natural organelles and facilitates the creation of more complex and multifaceted synthetic lifelike systems.