To begin, obtain a surfactant containing fluorinated, or a lipid mineral oil solution. Add cell-free protein synthesis or CFPS mixture to 500 microliters of the previously prepared oil in a 1.5 milliliter tube, and rub the tube vigorously on the tube rack 50 times to form fine droplets. For giant unilamellar vesicle, or GUV preparation, add 57 microliters of chloroform into a four milliliter glass vial, followed by 18 microliters of lipids, to a final concentration of eight millimolar.
Evaporate the chloroform under an argon flow for 15 minutes, and then under vacuum for one hour. Dissolve the resulting dry lipids in 1, 500 microliters of mineral oil to reach a final concentration of 400 micromolar. To obtain an interfacial lipid layer, slowly layer two 50 microliters of lipid mineral oil solution on top of 500 microliters of the outer solution, taken in a 1.5 milliliter tube.
Incubate at room temperature for 30 minutes to form a stable interfacial lipid layer. For the inner solution, add T7 RNA polymerase S30 extract, and DNA template into the cold pre inner solution. Mix 50 microliters of inner solution, and 500 microliters of lipid mineral oil, and vortex vigorously.
Leave the tube on ice for 10 minutes, and slowly add 20 microliters of this emulsion mixture to the top of the previously prepared oil phase in the 1.5 milliliter tube. Centrifuge at about 800 G for 10 minutes at four degrees celsius. And observe the formation of GUVs at the bottom of the tube.
After removing the upper oil phase, aspirate 30 microliters of GUVs from the bottom of the tube carefully. Add seven drops of sulfuric acid, and two drops of 50%hydrogen peroxide to the center of a cover slide, and incubate for 45 minutes. After rinsing thoroughly with ultrapure water, glue it to a cut 0.5 milliliters microfuge tube.
Cure under an ultraviolet lamp for 10 minutes to form a reaction chamber. Next, add 75 microliters of the small unilamellar vesicles, or SUV suspension to the preformed reaction chambers, and incubate it on a heat block at 37 degrees celsius for one minute. Then, add 150 microliters of supported lipid bilayer, or SLB buffer A to the reaction chamber for an additional two minute incubation.
Wash the chamber with hundred microliters of SLB buffer B prior to a buffer exchange to the S 30 buffer C, leaving 100 microliters of buffer inside the chamber to prevent the form's supported lipid bilayer from drying out. Using confocal laser scanning microscopy, the expression of green fluorescent protein and mCherry encapsulated in droplets was successfully captured. The fluorescence image of encapsulated CFPS within GUVs showed expression of super folder green fluorescent protein within the labeled lipid bilayer.
