Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

Protein producing synthetic cells offer a versatile platform for studying the origins of life as well as synthetic cell-based therapies for advanced drug delivery. This method is simple and affordable for RNA and protein expression and can be applied for onsite therapeutic protein production directly inside the body. The systems can be used for treating a wide range of diseases from metabolic diseases and protein replacements to cancer and possibly diabetes.

This multi-step protocol has a defined stopping point. When performing it for the first time, it is recommended to divide the work over several days. The protocol for synthetic cell production starts with the preparation of the cell-free S30-T7 lysate.

Next, the membrane lipids and cellular nutrient solution are prepared to simulate the inner environment and to support protein production. The final step is the preparation of the synthetic cells themselves. Prepare duplicate starter cultures in 100 milliliter Erlenmeyer flasks.

To each flask, add five milliliters of LB media supplemented with 50 micrograms per milliliter of ampicillin and inoculate with a single E.coli colony. Grow the culture overnight in a floor incubator shaker at 250 rpm and 37 degrees Celsius. Next, prepare two liter baffled Erlenmeyer flasks containing 500 milliliters of TB media supplemented with 50 micrograms per milliliter of ampicillin.

Inoculate each two liter flask with a five milliliter starter culture. Place the flasks in a shaker at 250 rpm and 37 degrees Celsius. Monitor the cultures periodically using a spectrophotometer and grow the cultures until OD600 between 0.8 and one.

To induce T7 RNA polymerase expression, add to each flask three milliliters of 100 millimolar IPTG to reach 0.6 millimolar. Continue growing the cultures until OD600 is approximately four. After the cultures reach the desired optical density, transfer the suspension from each Erlenmeyer flask into two 250 milliliter sterilized centrifuge tubes.

Centrifuge the tubes at 7, 000 times g for 10 minutes at four degrees Celsius. Discard the supernatant. Resuspend each pellet in 250 milliliters of cold S30 lysate buffer using a stirrer if desired.

Centrifuge at 7, 000 times g for 10 minutes at four degrees Celsius. Discard the supernatant and resuspend all the pellets together in 15 milliliters of cold S30 lysate buffer. Filter the suspension using gauze pads.

Before proceeding to the next step, precool the tips in 1.5 milliliter vials that will be needed for storing the lysate. Homogenize the suspension twice. Use a working pressure of 15, 000 PSI with an air pressure of four bar for cell breakage.

Collect the homogenate. Add 100 microliters of 0.1 molar DTT per 10 milliliters of the homogenized suspension. Centrifuge the suspension at 24, 700 times g at 30 minutes at four degrees Celsius.

DTT and chloroform are classified as irritant and harmful and should therefore be treated with care. Chloroform used later in the protocol must be used in an area with fume extraction. To preserve the lysate activity, perform the step quickly.

Place 200 microliter aliquots of supernatant in pre-cooled vials and immediately snap freeze them with liquid nitrogen. Store the vials at negative 80 degrees Celsius for future use. Separately dissolve POPC and cholesterol in chloroform each to a final concentration of 100 milligrams per milliliter.

Vortex each vial separately. Combine the components in a two milliliter glass vial. Add 50 microliters of the cholesterol-chloroform solution, 50 microliters of the POPC-chloroform solution, and 500 microliters of mineral oil.

Vortex the vial, then to evaporate the chloroform, heat it for about one hour at 80 degrees Celsius in a chemical hood. Before starting the synthetic cell production stage, prepare the outer, pre-inner, and feeding solutions as described in the manuscript. Then place 1.2 milliliters of the outer solution in a 15 milliliter tube and slowly add a layer of 500 microliters of lipids-in-oil solution from the first glass vial.

Incubate the tube at room temperature for 20 minutes. To finalize the inner solution preparation, mix on ice to a final volume of 100 microliters by adding S30-T7 lysate and DNA plasmid. Add 100 microliters of the inner solution with lysate and plasmid to the second two milliliter glass vial with 500 microliters of lipids-in-oil solution.

Pipette up and down vigorously for one minute and then vortex for another minute on level five. After incubating the resulting emulsion for 10 minutes on crushed ice, slowly add the emulsion on top of the oil phase in the 15 milliliter tube. Centrifuge the tube for 10 minutes at 100 times g and four degrees Celsius.

Then centrifuge for 10 minutes at 400 times g and four degrees Celsius. By the end of the centrifugation, a pellet should be observable at the bottom of the tube. To extract the pellet, first remove the excess oil layer.

Next, load a trimmed pipette tip with approximately 400 microliters of outer solution and use the pipette tip to collect the pellet, releasing the outer solution as the pipette tip passes through the oil phase. Wipe the pipette tip and transfer the pellet to a clean 1.5 milliliter tube. Centrifuge the pellet for 10 minutes at 1, 000 times g and four degrees Celsius.

Remove the supernatant and resuspend the pellet in 100 microliters of feeding solution. For protein expression, incubate the suspension for two hours at 37 degrees Celsius without shaking. Plasmids expressing the model protein sfGFP and Renilla luciferase were introduced into cell-free protein synthesis, CFPS bulk reactions, and synthetic cells.

Protein production was evaluated using several methods. Western blot analysis detected sfGFP His6 production in both CFPS bulk reactions and inside synthetic cells. Purified sfGFP His6 was used as a positive control.

A fluorescent microscope with a Brightfield and a GFP filter show synthetic cells that are producing sfGFP. Flow cytometry was used to analyze sfGFP producing synthetic cells. Calculation of the active synthetic cell population was based on the sfGFP fluorescence intensity threshold defined by the negative DNA sample represented by the red histogram.

Mean fluorescence intensity of sfGFP producing synthetic cells was calculated from the active synthetic cell population and normalized to the negative DNA sample. The diameter distributions of the active synthetic cells were calculated based on the sfGFP signal. Renilla luciferase activity was quantified with luminescence measurements.

This method is highly versatile and can be optimized for different target proteins by altering lysate origin, lipid composition and inner solution concentrations. FACS analysis of synthetic cells producing a fluorescent marker protein could be performed to provide quantitative value of the fraction of active synthetic cells. Western blot analysis would measure protein production.