  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.
