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

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.


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• Vortexer or emulsificator (optional) Note that Krytox concentration, lipid composition and buffer conditions can be adjusted as needed, but optimization of the parameters may be required to achieve the best possible GUV formation. For other tested lipid composition and buffer conditions see Table S1.

Procedure -Timing: 10 minutes
Note that the volumes are scalable to form 10 µL to hundreds of millilitre of GUVs.
The procedure described here is for the formation of 200 µl GUVs.

Tips for troubleshooting
If the efficiency of GUV formation is very low, please consider the following points: • Use SUVs within 48 h after formation.
using the Rhodamine 6G assay as described in Figure S5.
• Make sure that there is no osmotic mismatch between the encapsulated aqueous phase and the release buffer.
• Screen for optimal Krytox concentrations, lipid compositions and buffer conditions for your system.
• Perform FRAP measurements with the GUVs before the release (in the dropletstabilized state). If the diffusion coefficients are about 5 to 10 times lower than you would expect, it is possible that the SUVs have not fused at the droplet periphery. In this case, adjust the conditions (e.g. the Krytox concentration).
• To increase the yield, leave the dsGUVs at 4 • C over night before the release.
This will increase the yield.
• Increase the contact area between the aqueous release buffer and the droplet emulsion. If the droplets are too close to one another during the release, the GUVs may split or fuse during the process. Text S2, Figure S1: Choosing an appropriate lipid concentration Only if a sufficient amount of lipids is encapsulated inside the droplets, the GUVs can be released successfully. Therefore, we calculated the required lipid concentration as a function of GUV diameter. The lipid concentration has to be chosen such that the lipids can form a continuous bilayer at the droplet interface. It is important to consider that the surface-tovolume ratio decreases with increasing diameter. The required lipid concentration c Lip can be calculated as: where n Lip is the amount of lipid molecules and V Drop the volume of the droplet. The required number of lipid molecules, N Lip can be written as: where A Drop is the area of the droplet and d its diameter; A Head is the area occupied by a single lipid head group. The volume of the droplet V Drop is: Therefore, the required lipid concentration is inversely proportional to the droplet diameter and can be calculated as: With the Avogadro constant N A = 6.022 · 10 23 1 mol and assuming a lipid head group occupies an area of A Head = 0.7 nm 2 (according to values published earlier 1 ): The calculated lipid concentration as a function of GUV diameter is plotted in Figure S1.     Figure S4 are representative snapshots for a less efficient release (A) and a highly efficient release (B). By extrapolation, we estimate the number of GUVs per milliliter: 1 · 10 6 GUVs in the first and 2 · 10 7 GUVs in the latter case.
The release rate can be approximated by comparing the number of released GUVs to the number of dsGUVs. To produce 1 mL of released GUVs, we use 500 µL of the aqueous phase for droplet formation. By measuring the mean diameter of the dsGUVs, we extrapolate the total number of droplets (here: 4 · 10 7 ). This number is then divided by the number of released GUVs to estimate for the release rate -2.5 % in the first case, 50 % in the latter. Figure S5: Rhodamine 6G partitioning experiment   Table 1: Successfully tested combinations of buffer conditions and lipid compositions for the GUV production via the shaking method. The colour code indicates the release efficiency (gray: efficient (>15%); blue: medium (5%-15%); purple: less efficient (<5%) release) In all cases, the oil phase contained HFE-7500 fluorinated oil, 1.4 wt% PEG-based fluorosurfactant and 10.5 mM Krytox; 0.5 mol% Atto488-labelled DOPE or 1 mol% of LissRhod-PE was added to the lipid mixture for visualization purposes.     Video S1: Video protocol of the shaking method for GUV formation This video gives visual guidance to support first-time users of our shaking-method for the formation of GUVs. It follows the steps described in the manuscript (Figure 1 and Materials and Methods section) and the step-by-step protocol described above.

Video S3: Osmotic deflation of 'shaken' GUVs
This video shows a GUV produced by the shaking method that was osmotically deflated after formation. 4 Deflation leads to an excess membrane area and hence to budding and lipid tubulation.