Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil–surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

We calculate analytically the stochastic thermodynamic properties of an isothermal Brownian engine driven by a duo of time-periodic forces, including its Onsager coefficients, the stochastic work of each force, and the corresponding stochastic entropy production. We verify the relations between different operational regimes, maximum power, maximum efficiency, and minimum dissipation, and reproduce the signature features of the stochastic efficiency. All of these results are experimentally tested without adjustable parameters on a colloidal system.

Success in the bottom‐up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase‐separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light‐triggered uncaging of CMNB‐fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase‐separated vesicles from single‐phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

The bottom‐up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA‐encoded informational content are still in their infancy. Herein, droplets of DNA Y‐motifs are formed by liquid–liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y‐motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio‐temporally controlled DNA segregation in bulk as well as in cell‐sized water‐in‐oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice‐based theoretical model which mimics the interactions between the DNA Y‐motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom‐up assembled synthetic cells.