Membraneless Compartmentalization of Cell‐Free Transcription‐Translation by Polymer‐Assisted Liquid–Liquid Phase Separation

Living cells use liquid-liquid phase separation (LLPS) to compartmentalize metabolic functions into mesoscopic-sized droplets. Deciphering the mechanisms at play in LLPS is therefore critical to understanding the structuration and functions of cells at the subcellular level. Although observed and achieved to a significant degree of control in vivo, the reconstitution of LLPS integrating advanced biological functions, such as gene expression, has been so far limited in vitro. LLPS of cell-free transcription-translation (TXTL) reactions require multi-step experimental approaches that lack biomimetic and have relatively poor efficacy, thus limiting their usage in cell-free engineered systems such as synthetic cells. Here the polymer-assisted LLPS of TXTL reactions are reported as the single-pot one-step compartmentalization of a model complex metabolic system obtain without using solvents or surfactants. LLPS occurs by adding the biocompatible polymers poly(ethylene glycol), poly(vinyl alcohol), and dextran to a TXTL reaction, that remains highly active. These polymers serve as partitioning agents that localize TXTL in mesoscopic-sized droplets rich in dextran. Cytoplasmic and membrane-interacting proteins are synthesized preferentially inside these droplets, and localize either uniformly or preferentially at the interface, depending on their nature. The LLPS-TXTL system presented in this work is a step toward the design of synthetic membraneless active organelles.