Cell-Free Protein Synthesis System for Building Synthetic Cells

All our research focuses on cell-free synthetic biology. This involves bottom-up assembly of synthetic cells, functional and structural analysis of membrane proteins, protein engineering while incorporation of non-canonical amino acids and cell-free metabolic engineering. Currently, besides cell-free protein synthesis, microfluidic devices by microfabrication through soft lithography, development of biocompatible materials, and a general AI are the forefront technologies that drive the faster development of cell-free synthetic biology.

The configuration presented as a protocol establishes a straightforward yet robust system that is well-suited various micro-compartment environments, including the droplet GUVs as our base, that we showed in our representative results. In the future, we will focus on the development of tailored cell-free systems, which enable the assembly and integration of different modular cell mimicry systems towards the ultimate goal of building a minimal cell. To begin, obtain pre-cultured cells and wash them two times with 35 milliliters of pre-chilled S30 Buffer A.Pellet the cells and re-suspend them in S30 Buffer B.For disrupting the cells, transfer the cell suspension to a french press metal disruption chamber and secure it in the hydraulic platform with the safety lock.

Turn on the hydraulic pump and start the disruption. Control the outlet valve to collect the cell suspension in a new 50 milliliter tube, maintaining the pressure at 17, 000 pounds per square inch. Centrifuge the resulting lysate at 30, 000 G for 30 minutes at four degrees celsius and collect the supernatant.

Centrifuge again and incubate the collected supernatant with 0.3 volume of pre-incubation buffer with gentle shaking at 37 degrees celsius for 80 minutes. Dialyze the resulting runoff mixture for two hours against two liters of S30 Buffer C overnight at four degrees celsius. Centrifuge the dialyzed sample and aliquot the supernatant.

Flash-freeze the samples immediately in liquid nitrogen. After pelleting the cultured cells, re-suspend them in 30 milliliters of T7 Buffer A.Disrupt the cells in one pass through the french press at 15, 000 pounds per square inch and centrifuge the lysate to remove the cell debris. Add streptomycin sulfate drop by drop to the supernatant to reach a final concentration of 4%After a short incubation on ice, centrifuge the lysate as before.

Load the filtered samples onto a strong anion-exchange column. After washing the column, elute the sample with 10 column volumes of T7 Buffer C at a flow rate of 3 milliliters per minute. Add glycerol to reach a final concentration of 10%and subject the resulting mixture to ultra filtration to obtain a final concentration of three to four milligrams per milliliter.

To begin, obtain a surfactin-containing fluorinated or 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 250 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 800G 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 milliliter microfuge tube. Cure under an ultraviolet lamp for 10 minutes to form a reaction chamber.

Next, add 75 microliters of the small unilamellar vesicle, or SUV suspension, to the preformed reaction chambers and incubate 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 200 microliters of SLB Buffer B prior to a buffer exchange to S30 Buffer C, leaving 100 microliters of buffer inside the chamber to prevent the 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 fluorescent image of encapsulated CFPS within GUVs showed expression of super folder green fluorescent protein within the labeled lipid bilayer.