Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pKa, to drive liquid–liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pKa. We further show that pH‐triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi‐compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom‐up synthetic biology approach.

Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes.

A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substrate signaling, and enzyme cascade-mediated processing.

Many amphiphile-water mixtures will self-assemble into three-dimensional soft condensed structures known as inverse bicontinuous cubic phases. These structures are found in nature and have applications in nanotechnology. Here we show that by systematically varying amphiphile chain splay, we are able to control the relative stability of the inverse bicontinuous phases in a homologous series of monoglycerides in a predictable manner. In particular, we demonstrate that decreasing chain splay leads to the appearance of the primitive bicontinuous cubic phase while increasing chain splay reduces the channel size of the remaining two bicontinuous phases and tends to destabilize them with respect to the more curved inverse micellar and inverse hexagonal phases. These observations are consistent with a model in which the energetic stability of these phases is principally governed by the competing demands for homogeneous interfacial curvature and uniform chain packing and points to straightforward rules for engineering these self-assembling nanostructures.

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The spontaneous phase separation of peptide/nucleotide droplets in water produces membrane-free chemically organized micro-compartments that offer new opportunities for the construction of synthetic cells and development of protocell models of prebiotic organization. Certain small molecules can be sequestered into the droplet interior but the uptake mechanisms are unexplored. Using confocal fluorescence microscopy, 31 P NMR spectroscopy, fluorescence spectroscopy and lateral molecular force microscopy, we probe the molecular interactions associated with sequestration of the water-soluble fluorescent anionic dye 1-anilinonapthalene-8-sulphonic acid (ANS) into positively charged oligolysine/ ATP coacervate micro-droplets. Our results indicate that uptake of ANS proceeds initially through electrostatic interactions involving a ternary ANS/oligolysine/ATP complex, followed by a secondary mechanism based on non-polar interactions between ANS and ATP. We demonstrate that at very high levels of ANS the hybrid droplets develop a thin outer shell that is mechanically more compliant than the droplet interior, and acts as a quasi-membrane for restricting the influx of methylene blue. Our results suggest that understanding the mechanisms of molecular uptake into coacervate droplets could provide an important step towards the rational design of molecularly crowded microscale dispersions that display complex fluid behavior, compartment-mediated functionality and primitive aspects of synthetic cellularity.