This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.
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Method Article
Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer
* These authors contributed equally
In This Article
Summary
Abstract
The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA→RNA→protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.
Introduction
Synthetic cells are artificial cell-like particles, mimicking one or multiple functions of a living cell, such as the ability to divide, form membrane interactions, and synthesize proteins based on a genetic code1,2,3. Synthetic cells that enclose cell-free protein synthesis (CFPS) systems possess high modularity due to their ability to produce various proteins and RNA sequences following alterations in the DNA template. Presenting an attractive alternative to the current approaches of protein production, CFPS systems are based on cell lysate, purified components, or synthetic components and include all the transcription and translation machinery required for protein synthesis such as ribosomes, RNA polymerase, amino acids and energy sources (e.g., 3-phosphoglycerate and adenine triphosphate)4,5,6,7,8,9. The encapsulation of a CFPS system inside lipid vesicles enables the simple and efficient production of proteins without depending on a living cell10. Moreover, this platform allows synthesis of peptides that may degrade inside natural cells, production of proteins that are toxic to living cells, and modify proteins with non-natural amino acids11,12. Synthetic cells have been used as a model for research purposes investigating the minimal cell components required to enable cellular life from an evolutionary perspective1,13. Synthetic cells have also been used to build and implement genetic circuit and as models for directed evolution14,15,16. Other studies have focused on the ability of synthetic cells to mimic the biological activity of natural cells, aiming to replace damaged natural cells, such as beta cells in patients with diabetes17. Furthermore, the ability of these CFPS encapsulating synthetic cells to produce a variety of therapeutic proteins illustrates its potential to be incorporated into clinical use18.
Here we describe a bottom-up lab-scale protocol (Figure 1) for the production of RNA and protein-producing synthetic cells based on a CFPS system encapsulated in a lipid vesicle. This shows the potential use of synthetic cell platforms as novel drug delivery systems for the onsite production of a therapeutic protein drug in vivo19. Previous studies have investigated the optimization of the CFPS reaction and the cell lysate preparation processes4,8,20. Moreover, several techniques have been applied for cell-sized liposome preparation, such as microfluidic and polymer-based droplet stabilization methods21,22,23, which also differ in the liposomes' lipid composition24,25,26. In the presented protocol, synthetic cells are produced using a water-in-oil emulsion transfer method and the encapsulation process is carried out at low temperatures (<4 °C)5,10,24,27,28. These mild conditions have been found to be favorable for retaining the bio-functional integrity of the molecular machinery, namely ribosomes and proteins27,29,30. The lipid composition of the particles consists of both cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The first is found in all mammalian cell membranes and is essential for the stability, rigidity and permeability reduction of the membrane, and the latter mimics mammalian phospholipid composition11,13. The cellular transcription and translation molecular machinery are extracted from the BL21 (DE3) Escherichia coli (E. coli) strain, which is transformed with pAR1219 plasmid overexpressing T7 RNA polymerase to increase CFPS potency and protein synthesis. This system has been used to produce diagnostic and therapeutic proteins, with molecular weights of up to 66 kDa in vitro and in vivo19,31. The following protocol provides a simple and effective method for the production of the synthetic cell system, which can address a wide range of fundamental questions associated with protein synthesis in nature and can also be utilized for drug delivery applications.
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Protocol
NOTE: Illustration of the complete synthetic cells’ production protocol is presented in Figure 1. According to the user’s needs, the protein expression (section 3.2) and synthetic cell formation (section 4) parts of the protocol can also be carried out independently (with some adaptations).
1. Preparation of S30-T7 lysate
- Streak plate the E. coli BL21(DE3) bacteria transformed with the T7 RNA polymerase expressing pAR1219 plasmid on a LB-agar plate supplemented with 50 µg/mL ampicillin to obtain single colonies.
- Prepare a starter solution: Inoculate a single colony into 5 mL of LB-media supplemented with 50 µg/mL ampicillin in a 100 mL Erlenmeyer flask and grow overnight using a floor incubator shaker at 250 rpm and 37 °C. Prepare duplicates.
- Inoculate each 5 mL starter separately into 500 mL of TB media supplemented with 50 µg/mL ampicillin in a 2 L Erlenmeyer flask with baffles and grow it using a floor incubator shaker at 250 rpm and 37 °C until it reaches OD600 of 0.8-1. Monitor periodically using a spectrophotometer.
- Add 2-3 mL of 100 mM stock of IPTG (to reach 0.4 - 0.6 mM) for induction of T7 RNA polymerase expression and continue growing the culture until it reaches OD600≈4.
- Transfer the solution from each Erlenmeyer flask into two 250 mL sterilized centrifuge tubes.
- Centrifuge each at 7,000 x g for 10 min at 4 °C. Discard the supernatant.
NOTE: At this stage, the bacterial pellet can be stored at -20 °C for a few days before moving on to the next steps. - Re-suspend each pellet in 250 mL of cold (4 °C) S30 lysate buffer and centrifuge at 7,000 x g for 10 min at 4 °C.
NOTE: The S30 lysate buffer is used for maintaining protein stability after cell lysis is performed using the homogenizer in step 1.9. From this step forward, all steps until 1.12 should be carried out consecutively and rapidly.
NOTE: Before proceeding to the next step, pre-cool the tips and 1.5-milliliter vials that will be needed for storing the lysate - Discard the supernatant and re-suspend all pellets together in 15 mL of cold S30 lysate buffer. Filter the suspension using gauze pad.
NOTE: 15ml of solution is due to homogenizer min. volume. Should be noticed that bacteria concentration is also important. - Homogenize at a working pressure of 15,000 psi, with an air pressure of 4 bar (two passes) for cell breakage. Avoid solution dilution for a more concentrated and active lysate.
- Add 100 μL of 0.1 M DTT (CAUTION) per 10 mL of the homogenized suspension.
- Centrifuge the suspension at 24,700 x g for 30 min at 4 °C.
- Perform the following step quickly for preserving the lysate activity: divide the supernatant one-by-one into 200 μL aliquots in precooled 1.5 mL vials and immediately snap freeze them with liquid nitrogen. Store at -80 °C for further use.
CAUTION: DTT is classified as Irritant and Harmful and should therefore be treated with care.
2. Preparation of lipids in oil solution
- Dissolve POPC and cholesterol in chloroform (CAUTION) separately, each to a final concentration of 100 mg/mL. Vortex each vial separately.
- Combine the components in a 2mL glass vial: add 50 µL of POPC in chloroform, 50 µL of cholesterol in chloroform and 500 µL of mineral oil. For 100 µL of synthetic cells, 2 vials of lipids in oil are required.
- Vortex, and then heat for about 1 h at 80 °C in a chemical hood to evaporate the chloroform. Ensure that complete evaporation has occurred by following the specified time/conditions and monitoring the solution volume.
NOTE: The resulting lipid-in-oil solution can be stored at room temperature for up to two weeks. For improved results, it is recommended to use a fresh preparation before each experiment. Lipid and cholesterol ratios can be altered according to the desired membrane composition. A high concentration of cholesterol can lead to the formation of aggregates in the final synthetic cell solution.
CAUTION: Chloroform is classified as Irritant and Harmful and should therefore be treated with care and in areas with fume extraction.
3. Preparations of outer, pre-inner and feeding solutions
- Preparation of stock solutions
- Prepare the stock solutions listed in Table 2 using ultrapure water (UPW).
NOTE: Stock solutions should be prepared in advance and stored at -20 °C until further usage. Reagent 7 tends to form aggregates. Heating to 37 °C will reduce the aggregation. Slight aggregation will not affect the reaction significantly. Reagent 8 solution is milky and turbid.
- Prepare the stock solutions listed in Table 2 using ultrapure water (UPW).
- Outer solution
- Dissolve glucose in DNase/RNase-free H2O to a final concentration of 200 mM.
- For 100 µL of inner solution, prepare 1.6 mL of outer solution.
- Pre-inner solution
- Add reagents 1-14 according to the amounts and concentration listed in Table 2. For example, for a final synthetic cell volume of 100 µL, prepare 100 µL of inner solution.
NOTE: UPW should be added to complete the final required volume. At this stage, the mixture can be stored at 4 °C for a few hours.
- Add reagents 1-14 according to the amounts and concentration listed in Table 2. For example, for a final synthetic cell volume of 100 µL, prepare 100 µL of inner solution.
- Feeding solution
- Add all the reagents according to the amounts and concentration listed in Table 3.
NOTE: Use 1:1 ratio of feeding solution:inner solution. For a final synthetic cell volume of 100 µL, prepare 100 µL of feeding solution. It is recommended to prepare a small excess volume of outer, inner and feeding solution.
- Add all the reagents according to the amounts and concentration listed in Table 3.
4. Preparation of synthetic cells
NOTE: The following volumes are adjusted for the preparation of 100 µL of synthetic cells.
- Synthetic cells producing protein
- In a 15 mL tube, place 12 mL of the outer solution and slowly add, on top a layer, of 500 µL of lipids in oil solution. Incubate at room temperature for 20 min.
- To finalize the inner solution preparation: mix the inner solution ingredients on crushed ice to a final volume of 100 µL by thawing and adding S30-T7 Lysate (reagent 15) and DNA plasmid (reagent 16) to the stored mixture.
- To the second 2 mL glass vial with 500 µL of lipids in oil solution, add 100 µL of the inner solution. Pipette up and down vigorously for 1 minute and vortex for another minute on level five.
- Incubate for 10 min on crushed ice and slowly add the resulted emulsion on top of the oil phase in the 15 mL tube (from 4.1.1).
- Centrifuge for 10 min at 100 x g and 4 °C and then centrifuge for 10 min at 400 x g and 4 °C. By the end of the centrifugation, a pellet at the bottom of the tube should be observed.
NOTE: Using a swinging bucket centrifuge rotor is preferred here for acquiring a better coverage of a second layer of lipids during the water-in-oil droplets’ passage through the interphase. In case there is no observable pellet, centrifugation speed can be increased to 1000 x g. Otherwise, see the Discussion section referring to the specific gravity of the outer solution. - Extract the pellet.
- Remove excess oil layer.
- Use a trimmed pipette tip loaded with approximately 400 µL of outer solution to extract the pellet. Release the outer solution while passing through the oil phase in order to collect only the pellet in the aqueous phase.
- Wipe the tip after the extraction of the pellet to avoid transferring oil remains and transfer the pellet to a clean 1.5 mL tube.
- Centrifuge for 10 min at 1,000 x g and 4 °C, remove the supernatant and re-suspend the pellet in 100 µL of feeding solution (1:1 ratio of inner:feeding solutions).
NOTE: A fixed angle centrifuge rotor may be used here as well. - For protein expression, incubate for 2 hours at 37 °C without shaking.
NOTE: Optimal incubation time varies between different proteins. - Evaluate the produced protein amount using a suitable method according to the target protein properties.
- Recommended control groups
- Inner solution and lysate activity confirmation
- Prepare a complete inner solution (with DNA & S30-T7 lysate).
- Immediately incubate the reaction above using a floor incubator shaker at 250 rpm or a thermomixer at 1200 rpm, at a constant temperature of 37 °C for 2 h.
NOTE: Adjust the incubation time to match the synthetic cells incubation time.
- Synthetic cells
- Negative control group: Prepare the protocol presented in 4.1 with inner solution without DNA.
- Positive control: Prepare the protocol presented in 4.1 with inner solution containing a T7 plasmid encoding for a reporter gene.
NOTE: Add a positive control group comprised of a reporter gene, such as sfGFP, alongside the test groups to ensure the encapsulation efficiency step.
- Inner solution and lysate activity confirmation
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Results
We present a protocol for the preparation of synthetic cells by encapsulating a S30-T7 CFPS system based on BL21 E. coli inside lipid vesicles. A schematic description of the preparation process that includes an image of each stage is presented in Figure 2. The success of the synthetic cell preparation process is dependent on the appropriate performance of each stage and effected by different parameters. The protocol should be adjusted to accommodate the production of a specific pro...
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Discussion
This protocol introduces a simple and affordable method for the production of large quantities of protein-producing synthetic cells. The yield of active cells is dependent on careful and accurate execution of the protocol with emphasis on several critical steps. In the lysate preparation section of this method, it is essential to reach the appropriate bacteria density before cell lysis to achieve a sufficient amount of proteins in the bacterial lysate. Second, the lysis process should be performed at 4 °C and the ly...
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Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by ERC-STG-2015-680242.
The authors also acknowledge the support of the Technion Integrated Cancer Center (TICC); the Russell Berrie Nanotechnology Institute; the Lorry I. Lokey Interdisciplinary Center for Life Sciences & Engineering; the Israel Ministry of Economy for a Kamin Grant (52752); the Israel Ministry of Science Technology and Space – Office of the Chief Scientist (3-11878); the Israel Science Foundation (1778/13, 1421/17); the Israel Cancer Association (2015-0116); the German-Israeli Foundation for Scientific Research and Development for a GIF Young grant (I-2328-1139.10/2012); the European Union FP-7 IRG Program for a Career Integration Grant (908049); the Phospholipid Research Center Grant; a Rosenblatt Foundation for cancer research, a Mallat Family Foundation Grant; and the Unger Family Foundation. A. Schroeder acknowledges Alon and Taub Fellowships. O. Adir acknowledges the Sherman and Gutwirth fellowships. G. Chen acknowledges the Sherman Fellowship. N. Krinsky acknowledges the Baroness Ariane de Rothschild Women Doctoral Program from the Rothschild Caesarea Foundation.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| A. Reagents required for step 1 (S30-T7 lysate preparation) | |||
| E.coli BL21 (DE3) | NEB | C2527 | E.coli BL21 (DE3). |
| pAR1219 | Sigma | T2076 | TargeTron vector for transformation. |
| Stock solution of 50 mg/mL Ampicillin | Sigma | A9518 | Stored at -20 °C. |
| 10 g/L Bacto-tryptone | BD Bioscience | 211705 | For preparation of Luria Bertani (LB) agar (1.5%) plate. |
| 10 g/L Sodium chloride (NaCl) | Bio-Lab | 19030591 | |
| 5 g/L Bacto-Yeast extract | BD Bioscience | 212750 | |
| 15 g/L Agar agar purified | Merck | 1.01614.5007 | |
| 50 µg/mL Ampicillin | Sigma | A9518 | |
| 10 g/L Bacto-tryptone | BD Bioscience | 211705 | For preparation of Luria Bertani (LB) media (20 mL). |
| 10 g/L Sodium chloride (NaCl) | Bio-Lab | 19030591 | |
| 5 g/L Bacto-Yeast extract | BD Bioscience | 212750 | |
| 50 µg/mL Ampicillin | Sigma | A9518 | |
| 12 g/L Bacto-tryptone | BD Bioscience | 211705 | For preparation of Terrific Broth (TB) media (1 L). |
| 24 g/L Bacto-Yeast extract | BD Bioscience | 212750 | |
| 4% (v/v) Glycerol anhydrous | Bio-Lab | 7120501 | |
| 2.32 g/L K2HPO4 | Spectrum chemical | P1383 | |
| 12.54 g/L KH2PO4 | Spectrum chemical | P1380 | |
| 50 µg/mL Ampicillin | Sigma | A9518 | |
| Stock solution of 100 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) | INALCO | INA-1758-1400 | Filtered using 0.2 µm hydrophilic PVDF syringe filter. |
| Stock solution of 0.1 M dithiothreitol (DTT) | TCI | D1071 | Filtered using 0.2 µm hydrophilic PVDF syringe filter. |
| 10 mM Tris-acetate at pH = 7.4 | Sigma | T1503 | S30 lysate buffer (1.5 L) |
| 14 mM magnesium acetate | Merck | 1.05819.0250 | |
| 60 mM potassium acetate | Carlo Erba | 470147 | |
| 1 mM DTT | TCI | D1071 | |
| 0.5 mL/L 2-mercaptoethanol | Sigma | M6250 | |
| Equipment required for step 1 | |||
| 100 mL sterilized Erlenmeyer flasks | Thermo Scientific | 50-154-2846 | 2 flasks |
| 2 L sterilized Erlenmeyer flasks with baffles | KIMAX-KIMBLE | 25630 | 2 flasks |
| Floor incubator shaker | MRC | TOU-120-2 | Laboratory shaker incubator 450x450mm, 400rpm, 70 °C |
| Centrifuge | Thermo Scientific | 75004270 | (75003340) - Fiberlite F10-6 x 100 LEX Fixed-Angle Rotor. Should enable at least 13,000 x g. * Pre-cooled to 4 °C. |
| High pressure homogenizer | AVESTIN | EmulsiFlex-C3 | Pre-cooled to 4 °C. |
| -80oC freezer | SO-LOW | U85-18 | |
| Sterilized 1.5 mL plastic tubes | Eppendorf | 30120086 | Preferably pre-cooled to -20 °C. |
| Spectrophotometer | TECAN | IN-MNANO | Infinite M200 pro |
| 96-well transparent plate | Thermo Scientific | 167008 | |
| Sterilized graduated cylinder | Corning | ||
| Sterilized centrifuge tubes | Eppendorf | 30120086 | Preferably pre-cooled to -20 °C. |
| Sterilized pipette tips | Corning | Preferably pre-cooled to -20 °C. | |
| Crushed ice bucket | Bel-Art | M18848-4001 | |
| Small liquid nitrogen tank | NALGENE | 4150-4000 | |
| B. Reagents required for step 3 (lipids in oil solution preparation): | |||
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) | Lipoid | 556400 | Powder |
| Cholesterol | Sigma | C8667 | Powder |
| Chloroform | Bio-Lab | 3082301 | |
| Mineral oil | Sigma | M5904 | Light oil |
| Equipment required for step 2 | |||
| Vortex mixer | Scientific industries | SI-0256 | |
| Heating block | TECHNE | FDB03AD | Pre-heated to 80 °C. Should enable controlled temperature. |
| 2 mL screw neck glass vials | CSI Analytical Innovations | VT009M-1232 | For a larger scale, use 50 mL falcons and evaporate the chloroform using rotary evaporator. |
| 9mm Screw Cap | CSI Analytical Innovations | C395R-09LC | |
| C. Reagents required for step 3 (inner and feeding reaction mixtures): | |||
| HEPES | Spectrum | H1089 | 1 M HEPES-KOH (pH = 8) - pH buffer |
| Potassium hydroxide (KOH) | Frutarom | 55290 | |
| 1 M Magnesium acetate | Merck | 1.05819.0250 | Co-factor and negative charge stabilizor. |
| 1 M Potassium acetate | Carlo Erba | 470147 | Negative charge stabilizor. |
| 5.2 M Ammonium acetate | Merck | 1.01116.1000 | Stabilizes negative charge. |
| 50% (w/v) Polyethylene glycol 6000 (PEG) | Merck | 8.07491.1000 | Increases the concentration of the macromolecules. |
| 0.5 M 3-phosphoglycerate (3-PGA) | Sigma | P8877 | Secondary energy source. |
| 50 mM Amino acids mixture I | Sigma | LAA21-1KT | Amino acids additive. Contains: 50 mM of each of the following 17 natural amino acids - alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and valine. |
| 50 mM Amino acids mixture II | Sigma | LAA21-1KT | Amino acids additive. Contains: 50 mM of each of the following 3 natural amino acids - tryptophan, phenylalanine, and tyrosine. |
| 100 mM Adenine triphosphate (ATP) | Sigma | A3377 | Nucleotides & energy source. |
| 50 mM Guanidine triphosphate (GTP) | Sigma | G8877 | Nucleotides & energy source. |
| 100 mM Uridine triphosphate (UTP) | ACROS ORGANICS | 226310010 | Nucleotides additive. |
| 100 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) | INALCO | INA-1758-1400 | Genes expression induction. |
| 2 M Sucrose | J.T. Baker | 1933078 | Generating a density gradient. |
| 2 M Glucose | Sigma | 16301 | Generating a density gradient. |
| H2O UltraPure Water (UPW) | Bio-Lab | 2321777500 | DNase & RNase free |
| S30-T7 lysate | _ | _ | Prepared at step 1. Source of transcription & translation components. Store at -80 °c, thaw on crashed ice just before usage. |
| Stock of DNA plasmid of choice | _ | _ | Contains the sequence for the requested protein. Under T7 promotor |
| D. Equipment required for step 4 (synthetic cells preparation) | |||
| Floor incubator shaker or Thermomixer | MRC | TOU-120-2 | Laboratory shaker incubator 450x450mm, 400rpm, 70 °C |
| PHMT Grant Bio | PSC18 | Thermomixer | |
| Centrifuge | Thermo Scientific | 75004270 | (75003629) - TX-400 4 x 400mL Swinging Bucket Rotor. Suited for 15 mL sized tubes. Preferably swinging buckets. Should enable at least 1000 x g. Pre-cooled to 4 °C. |
| Table centrifuge | Thermo Scientific | 75002420 | (75003424) - 24 x 1.5/2.0mL rotor with ClickSeal. Suited for Eppendorf vials. Pre-cooled to 4 °C. |
| Vortex mixer | Scientific industries | SI-0256 | |
| Crushed ice bucket | Bel-Art | M18848-4001 | |
| 2 mL screw neck glass vials | CSI Analytical Innovations | VT009M-1232 | |
| Sterile 15 mL plastic tubes | Thermo Scientific | 339651 | |
| Sterilized 1.5 mL plastic tubes | Eppendorf | 30120086 | |
| Sterilized pipette tips | Corning | Sterilized by autoclave. |
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