One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells

Abstract

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.

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport

“…Accordingly, manipulating the charge on the vesicles could lead to more stable multiple compartments. 12 Another strategy to create stable multiple compartments is achieved by including PEG 8000 in the buffer to the DOPC:DOPG mixture lipid lm (Video S4 †). However, the efficiency of encapsulating vesicles in GUVs is low compared to that of microuidic techniques.…”

“…Another hypothesis might be that some proportion (close to 5-10%) of the vesicles are multilamellar, as observed for other methods as well. 12 Also, we present the data for different individual GUVs measured at both pH 5 (n ¼ 8) and pH 7 (n ¼ 9) and corresponding I in /I out changing at different rates conrming varying protein reconstitution in different GUVs (Fig. S13 †).…”

“…2,8 Several solvent-displacement techniques have been used for giant vesicle preparation with a variety of complex lipids under physiological salt conditions. [9][10][11][12] Further microuidic technologies allow for microcompartment formation in vesicles for synthetic biology applications using the solvent-displacement method. 13,14 However these techniques are incompatible to investigate surface properties as organic solvents cannot be removed effectively.…”

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

“…Vesicles were usually utilized for fabricating artificial cell models and applied for understanding biological cells . It was reported that the compartmental vesicle was applied as the hypothetical precursor of cell at the origin of life . Correspondingly, preparation size‐controllable monodisperse vesosomes should be the first problem to be solved for fabricating artificial cells.…”

“…[219][220][221] It was reported that the compartmental vesicle was applied as the hypothetical precursor of cell at the origin of life. [222][223][224] Correspondingly, preparation size-controllable monodisperse vesosomes should be the first problem to be solved for fabricating artificial cells. Microfluidic devicebased methods may help to achieve this goal by taking advantages of the ability to control vesicular structures.…”