Cell-Free Protein Synthesis System for Building Synthetic Cells

1. Extract preparation

1. Streak the E. coli BL21 (DE3) strain from a glycerol stock onto a Luria Bertani (LB) agar plate and incubate for at least 15 h at 37 °C.
2. Prepare an overnight preculture by inoculating a single colony from the freshly prepared LB plate into a 50 mL flask of Luria Bertani (LB) medium.
3. Inoculate 5 mL of preculture into 500 mL of 2xYTPG medium in a 3 L baffled Erlenmeyer flask. Grow it at 37 °C with vigorous shaking (between 220 rpm and 250 rpm) and harvest the cells when they reach the mid-log phase (OD₆₀₀ ~ 3). Then, place the cell cultures in cold icy water for 10 min and centrifuge at 8,000 × g for 15 min at 4 °C.
   NOTE: In this step, the glucose in 2xYTPG medium can be omitted for specific applications^9,12.
4. Resuspend the resulting cell pellets in 35 mL of prechilled S30 buffer A, followed by centrifugation at 8,000 × g for 15 min at 4 °C. Discard the supernatant.
5. Repeat step 1.4 twice.
   NOTE: The cell pellets can be stored at -80 °C if not used immediately.
6. Resuspend the final pellets in S30 buffer B (e.g., use 1.1 mL of S30 buffer B per 1 g of cell pellet) and disrupt the cells by one pass through French Press at 17,000 psi.
   1. For using the French Press, follow the user manual and transfer the cell suspension to the French press metal disruption chamber, ensuring that all components are assembled and the bottom valve of the disruption chamber is closed.
   2. Transfer the assembled disruption chamber to the hydraulic platform and secure the safety lock.
   3. Turn on the hydraulic pump and start the disruption.
   4. Control the outlet valve to enable the cell suspension to flow out of the outlet tube into a new 50 mL tube. Adjust the flow speed to ensure efficient disruption, ideally one drop at a time. Maintain the pressure above the lower limit of 15,000 psi.
7. Centrifuge the resulting lysate at 30,000 × g for 30 min at 4 °C and collect the supernatant. Repeat the centrifugation once and collect the supernatant. Add 0.3 volume of preincubation buffer into the collected supernatant and incubate with gentle shaking at 37 °C for 80 min.
   NOTE: The use of preincubation buffer could increase the final yield though it has been reported that an empty run-off (without preincubation buffer) could also improve the efficiency^37,38, which depends on the corresponding source strain.
8. Dialyze the resulting run off mixture for 2 h against 2 L of S30 buffer C, exchange the dialysis buffer once with 2 L of fresh S30 buffer C, and dialyze overnight at 4 °C³⁹.
   NOTE: The second step of overnight dialysis can be shortened to approximately 3 h.
9. Collect the dialyzed sample and centrifuge at 30,000 × g for 30 min at 4 °C. Collect the supernatant and aliquot into appropriate volumes and flash freeze immediately in liquid nitrogen.
   NOTE: The frozen sample can be stored at -80 °C at least for 6 months to 1 year without loss of efficiency.

2. T7 RNA polymerase

1. Transform pAR1219 plasmid into BL21(DE3) star E. coli competent cells⁴⁰.
2. Inoculate 10 mL of an overnight culture into 1 L of LB medium (containing 100 µg/mL ampicillin). Grow the cells at 37 °C until OD₆₀₀ reaches 0.6-0.8.
3. Start the induction by adding a final concentration of 1 mM IPTG. Induce the cells for an additional 5 h and harvest by centrifugation at 8,000 × g for 15 min at 4 °C. Store the cell pellets at -80 °C for up to several weeks.
4. Resuspend the cell pellets in 30 mL of T7 buffer A and disrupt the cells by one pass through the French Press at 15,000 psi. Remove the cell debris by centrifugation at 20,000 × g for 30 min at 4 °C.
5. Add streptomycin sulfate drop by drop to the supernatant from the previous step, reaching a final concentration of 4% (w/v). After a short incubation on ice, centrifuge at 20,000 × g for 30 min at 4 °C.
6. Filter the supernatant through a 0.45 µm filter membrane and load the filtered sample onto a strong anion exchange column (column volume of 40 mL), which was preequilibrated with 10 column volumes of T7 buffer B, via an automated liquid chromatograph system.
7. Wash the loaded column with 50 column volumes of T7 buffer B and elute the sample with 10 column volumes of low salt (50 mM NaCl) and high salt (500 mM) T7 buffer C mixtures, establishing a linear concentration gradient of NaCl from 50 to 500 mM at a flow rate of ~3 mL/min⁴¹.
8. Collect the peak fractions and analyze by SDS-PAGE.
   NOTE: T7 polymerase exhibits a predominate band at ~100 kDa, while significant amounts of impurities still appear on the SDS-PAGE gel.
9. Pool the fractions containing T7 polymerase and dialyze against 2 L of T7 buffer C overnight.
10. Add glycerol to reach a final concentration of 10% and concentrate the resulting mixture to a final concentration of 3-4 mg/mL by ultrafiltration⁴².
    NOTE: If precipitation appears during the concentration process, stop immediately and remove the precipitates by centrifugation.
11. Adjust the glycerol concentration to 50%. Aliquot and flash freeze the sample with liquid nitrogen. Store at −80 °C for a longer period.
    NOTE: A small aliquot could also be stored at -20 °C for at least 1 month without loss of efficiency.

3. Buffer preparation

1. Prepare buffers as indicated in Table 1 a day before usage.

4. Template design and preparation

1. Clone the gene of interest into a T7 promoter-based vector or generate a linear PCR product containing the gene of interest.
   NOTE: See the discussion section for design principles.
2. Prepare the plasmid from an overnight culture using a plasmid extraction kit.
   NOTE: We recommend selecting a plasmid kit that includes an isopropanol precipitation step followed by a washing procedure with 70% ethanol.
3. Dissolve the dried DNA in a small volume of ultrapure water to a concentration between 200 µg/mL and 500 µg/mL, as determined by a micro volume spectrophotometer.
4. Optional: Directly express proteins from linear PCR products.
   NOTE: In this case, a PCR purification step is needed.

5. Mg²⁺ and K⁺ optimization

1. Prepare a master mix for Mg²⁺ concentration screening as indicated in Table 2, or for K⁺ concentration screening as indicated in Table 3.
2. Transfer the master mix into individual microfuge tubes or a V-shaped 96-well plate.
3. Pipette the exact volume of Mg²⁺ or K⁺ stock solutions into individual microfuge tubes or the V-shaped 96-well plate and complete the CFPS reactions with ultrapure water. Incubate the reactions for at least 2 h.
   NOTE: If using a V-shaped 96-well plate, seal the plate with a plastic cover to prevent evaporation.
4. Take out 2 µL of the reaction mixture and transfer into a black 96-well plate for fluorescence measurements by a plate reader.
   NOTE: We use a fluorescent plate reader with the following setup: excitation/emission: 485/528, Gain: 50, Read height: 7.00 mm. However, one would need to tune their plate reader to generate a specific calibration curve using purified fluorescent proteins.

6. Encapsulation

1. Droplet
   1. Prepare a surfactant-containing fluorinated oil (2% PFPE-PEG in HFE7500) or a lipid-mineral oil solution by dissolving lipid in mineral oil (refer to step 6.2.1)
   2. Prepare a total volume of 100 µL of CFPS reaction by combining the corresponding reagents 2-18 from Table 4. Add CFPS reaction to 500 µL of the previously prepared oil in a 1.5 mL tube and then, rub the tube vigorously on the tube rack 50x to form fine (water-in-oil) droplets. Incubate the tube at 30 °C to perform the reaction.
      NOTE: Simple microfluidics chips could also be used to generate homogenous droplets as well⁴³.
2. Giant unilamellar vesicles (GUVs)
   1. Preparing the lipid-mineral oil solution
      1. Add 57 µL of chloroform into a 4 mL glass vial and then add 18 µL of 25 mg/mL 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC) lipids to form the chloroform lipid solution, achieving a final concentration of 8 mM (use other lipids with similar final concentrations).
         NOTE: This mixing step was to ensure homogeneous mixing of different lipids.
      2. Evaporate the chloroform under an argon flow for 15 min, and then, further evaporate under vacuum for 1 h.
      3. Dissolve the resulting dry lipids in 1,500 µL of mineral oil, reaching a final concentration of 400 µM. Incubate the mixture overnight at room temperature.
   2. Forming an interfacial lipid layer
      1. Add 500 µL of the outer solution (see Table 5) to a 1.5 mL tube, and slowly layer 250 µL of lipid-mineral oil solution on top of the outer solution. Incubate at room temperature for 30 min to form a stable interfacial lipid layer.
   3. Preparation of the inner solution
      1. Mix all the reagents in Table 6 to form the preinner solution and keep it on ice until use. Complete the inner solution by adding T7RNAP, S30 extract, and DNA template into the preinner solution as reagents 16-18 listed in Table 4.
         NOTE: Increasing encapsulated sucrose concentration above 250 mM might inhibit cell-free translation⁴⁴.
   4. Formation of GUVs
      1. Add 50 µL of inner solution to a 1.5 mL new tube containing 500 µL of lipid-mineral oil, pipette up and down rapidly, and vortex vigorously.
      2. Leave the tube on ice for 10 min and slowly add 20 µL of the emulsion mixture to the top of the oil phase in the 1.5 mL tube from step 6.2.2.
      3. Centrifuge at 800 × g (use a centrifugation speed ranging from 100 × g to 1000 × g) for 10 min at 4 °C and observe the formation of GUVs at the bottom of the tube.
         NOTE: A higher specific centrifugation speed could be used; however, one would consider that the centrifugation speed could influence the size of formed GUVs⁴⁵.
      4. Remove the upper oil phase.
      5. Aspirate 30 µL of GUVs at the bottom of the tube carefully. Incubate the collected GUVs at 30 °C.
         NOTE: The optimal incubation temperature varies for different proteins.
      6. Use Confocal Laser Scanning Microscopy (LSM) to monitor the expression of fluorescent proteins.
3. Supported lipid bilayer (SLB)
   1. Preparation of SLB chambers
      1. Piranha-clean 24 x 24 mm #1.5 coverslips by adding seven drops of sulfuric acid and two drops of 50% hydrogen peroxide to the center of each cover slide. Incubate the reactants on the coverslips for at least 45 min; rinse thoroughly with ultrapure water.
      2. Assemble the reconstitution chamber by attaching a cut 0.5 mL microfuge tube onto the cleaned coverslips using optical glue cured under an ultraviolet lamp (365 nm) for 10 min to form a reaction chamber.
   2. SLB formation
      1. Dissolve 80 mol% 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 19.95 mol% 1,2-dioleoyl-sn-glycerol-3-phospho-L-serine (DOPS), and 0.05 mol% Atto488-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine = DOPE), mix in chloroform, dry under mild nitrogen flow and vacuum for 1 h to remove the solvent completely.
      2. Rehydrate the dried lipid film in SLB buffer A, resulting in a final lipid concentration of 4 mg/mL.
      3. Vortex and sonicate the resulting samples until they become clear.
      4. Dilute 4 mg/mL of SUVs with 130 µL of SLB buffer A, transfer 75 µL of the suspension to the preformed reaction chambers, and incubate it on a heat block at 37 °C for 1 min. Add 150 µL of SLB buffer A to the reaction chamber for further incubation for 2 min.
      5. Wash the chamber with 2 mL of SLB buffer B (SLB buffer A without MgCl₂) prior to a buffer exchange to the S30 buffer C with 0.4% (w/v) BSA for CFPS reactions, leaving 100 µL of buffer inside the chamber to prevent the formed SLB from drying out.
      6. Remove the residual S30 buffer C and add the CFPS reaction mixture into the SLB chamber carefully. Incubate the chamber at 30 °C and monitor the expression under a confocal laser scanning microscope.