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    Abstract The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription–translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells. 
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    Liquid-liquid phase separation (LLPS) is important to control a wide range of reactions from gene expression to protein degradation in a cell-sized space. To bring a better understanding of the compatibility of such phase-separated structures with protein synthesis, we study emergent LLPS in a cell-free transcription-translation (TXTL) reaction. When the TXTL reaction composed of many proteins is concentrated, the uniformly mixed state becomes unstable, and membrane-less phases form spontaneously. This LLPS droplet formation is induced when the TXTL reaction is enclosed in water-in-oil emulsion droplets, in which water evaporates from the surface. As the emulsion droplets shrink, smaller LLPS droplets appear inside the emulsion droplets and coalesce into large phase-separated domains that partition the localization of synthesized reporter proteins. The presence of PEG in the TXTL reaction is important not only for versatile cell-free protein synthesis but also for the formation of two large domains capable of protein partitioning. Our results may shed light on the dynamic interplay of LLPS formation and cell-free protein synthesis toward the construction of synthetic organelles. 
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