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1. Preparation of Cell-free Reaction Components
- General reaction components
- Prepare working stocks of Mg-glutamate and K-glutamate in 50 mL tubes with sterile DI (de-ionized) water at concentrations of 100 mM and 2000 mM, respectively.
- Prepare a working stock of 50% (w/v) polyethylene glycol (PEG)-8000 by adding 100 mL of sterile DI water to a 250 mL beaker. Place a small magnetic stirrer into the beaker. Weigh out 50 g of PEG-8000 and add to the 100 mL of water in the beaker.
- Place the beaker on a heated stir plate set to 100 °C and stir at 250 rpm until PEG-8000 is in solution. Allow PEG-8000 liquid mixture to cool before transferring to 50 mL tubes.
- Energy solution master mix
- Prepare a 5 M solution of KOH by adding 140 g of KOH pellets to 500 mL of sterile DI water.
CAUTION: Prepare this solution in a chemical hood and wear PPE when handling strong bases. The solution will become hot so the bottle cap must be loose to prevent pressure build-up. Allow the KOH solution to reach RT before using. - Prepare a 1750 mM HEPES-KOH buffer by adding 20.85 g of HEPES to a 100 mL bottle. Slowly add sterile DI water until the volume reaches 40 mL. Vortex bottle to dissolve the HEPES. Use the 5 M KOH solution to adjust the pH to 8.0 and then bring the solution volume to 50 mL.
CAUTION: KOH should be handled with proper PPE when adjusting the pH. - Prepare the remaining 10x energy master mix stock components at the concentrations indicated in Table 1 in sterile DI water and place each stock on ice. Thaw the 100 mM ATP, GTP, CTP, and UTP stocks at RT and place on ice.
NOTE: A thermomixer set to 37 °C and 350 rpm can be used to dissolve reagents into the solution if necessary. Do not overheat or leave reagents on the thermomixer for an extended period of time. - In a 15 mL tube, add each energy solution master mix component in accordance with the order and volume specified in Table 1. Vortex the solution after each component is added. This will make 5 mL of 10x energy solution master mix.
- Divide the 10x energy solution master mix into 200 μL aliquots in 2 mL tubes. Flash-freeze each aliquot. Immediately place them into the -80 °C freezer until use.
- Amino acid master mix
- To prepare a fresh 4x amino acid master mix, begin by thawing each amino acid stock at RT and then placing each on ice. Use a vortex and/or thermomixer set at 37 °C and 350 rpm to ensure all amino acid stocks are fully dissolved.
NOTE: Cysteine may not fully dissolve; it can be added to the amino acid master mix as a suspension. Do not overheat or leave reagents on the thermomixer for an extended period of time. - In a 15 mL tube, add the appropriate volume of amino acids to sterile DI water so that the final concentration of each is 8 mM in the following order: ALA, ARG, ASN, ASP, GLN, GLU, GLY, HIS, IIE, LYS, MET, PHE, PRO, SER, THR, VAL, TRP, TYR, LEU, and CYS. After adding each amino acid, vortex the master mix solution. The volumes listed in Table 1 will make up 2.4 mL of amino acid master mix.
- Divide the 4x amino acid master mix into 200 μL aliquots in 2 mL tubes. Flash-freeze each aliquot. Immediately place into a freezer at -80 °C until use.
- Production of reaction-ready plasmid DNA template
NOTE: Cell-free protein expression in this system has been optimized using the super folder green fluorescent protein (GFP) expression vector T7-pJL1-sfGFP (Table of Materials). It is recommended to use this plasmid as a control for cell-free reaction efficiency and the pJL1 backbone for the cloning and expression of other protein sequences. Other plasmid DNA templates can be used; however, it is important to note that transcription is controlled by a T7 promoter sequence and the presence of T7 RNA polymerase. A simple protocol for the large-scale production of any plasmid DNA template from transformed E. coli is described below.- Purify the desired vector using a plasmid purification kit as per the manufacturer’s instructions (Table of Materials).NOTE: Concentrating the plasmid DNA template as much as possible is recommended to meet the tight volume constraints of the cell-free reaction. In general, aim for a 750−1500 ng/μL working stock.
2. Performing Cell-free Protein Expression Reactions Using V. natriegens Crude Extract
- Cell-free protein expression using plasmid or linear DNA template
- Remove 10x energy solution master mix and 4x amino acid master mix aliquots from the -80 °C freezer, thaw at RT, and place on ice. Remove the T7 RNA polymerase and RNase inhibitor stocks from the -20 °C freezer and place them on ice. Thaw the DNA template at RT and place on ice.
- Prepare a cell-free reaction master mix as per Table 2 by adding each component in the following order to a 2 mL tube on ice: amino acid master mix, energy solution master mix, Mg-glutamate, K-glutamate, DNA template, PEG-8000, T7 RNA polymerase, and RNase inhibitor. Gently flick the tube after each addition to the master mix.
NOTE: If a linear template is to be used for cell-free protein expression, add 5−10x more material as compared to the plasmid template to obtain appreciable yields of protein. - Remove V. natriegens crude cell lysate from the -80 °C freezers and place on ice for 10−20 min until thawed. Add the appropriate volume of crude cell extract to the cell-free reaction master mix as per Table 2 and gently mix by flicking or pipetting up and down.
- End-point cell-free protein expression using a thermocycler
- Pipette 10 μL of the cell-free reaction master mix to the bottom of a 96- or 384-well PCR plate. In between each transfer to the PCR plate, mix the master mix by flicking the tube gently.
NOTE: Cell-free reaction master mix should be well mixed at all times to maximize reaction reproducibility and cell-free protein expression in all samples.
- Briefly centrifuge the plate at 1,000 x g for 10 s to pool any master mix that may have become stuck on the sides of the wells. Seal the wells with a plate adhesive to prevent evaporation, and then place the PCR plate into a thermocycler set at 26 °C with a heated lid set at 105 °C.
NOTE: The even heat distribution and heated lid of a thermocycler greatly improve protein expression yields. - Incubate the cell-free reactions for a minimum of 3 h. After incubation, expressed proteins can be purified, quantified, and used for downstream processes.
NOTE: Expressed proteins can be directly quantified in the cell-free reaction using a method of the user’s choice. For example, fluorescent proteins can be quantified using an external standard curve, or radioactivity can be measured if using a radiolabeled amino acid in the cell-free reaction. UV-visible spectroscopy or total protein assays are generally not recommended for directly measuring protein production in cell-free reactions without an initial purification.
Table 1: Reagents for the preparation of 5 mL of 10x energy solution master mix and 2.4 mL of 4x amino acid master mix.
| Preparation of 10x Energy Solution Master Mix | |
|---|
| Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) | Final Volume (µL) |
| HEPES-KOH pH 8 | 1750 | 500 | 1428.57 | 5000 |
| ATP | 100 | 15 | 750.00 |
| GTP | 100 | 15 | 750.00 |
| CTP | 100 | 9 | 450.00 |
| UTP | 100 | 9 | 450.00 |
| tRNA from E. coli MRE 600 (mg/mL)* | 100 | 2 | 100.00 |
| Coenzyme A Hydrate | 200 | 2.6 | 65.00 |
| NAD | 200 | 3.3 | 82.50 |
| cAMP | 650 | 7.5 | 57.69 |
| Folinic Acid | 100 | 0.7 | 35.00 |
| Spermidine | 1600 | 10 | 31.25 |
| 3-PGA | 2000 | 300 | 750.00 |
| Sterile Deionized Water | | | 49.99 |
| | Preparation of 4x Amino Acid Master Mix |
| Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) | Final Volume (µL) |
| ALA | 168 | 8 | 114.3 | 2400 |
| ARG | 168 | 8 | 114.3 |
| ASN | 168 | 8 | 114.3 |
| ASP | 168 | 8 | 114.3 |
| GLN | 168 | 8 | 114.3 |
| GLU | 168 | 8 | 114.3 |
| GLY | 168 | 8 | 114.3 |
| HIS | 168 | 8 | 114.3 |
| IIE | 168 | 8 | 114.3 |
| LYS | 168 | 8 | 114.3 |
| MET | 168 | 8 | 114.3 |
| PHE | 168 | 8 | 114.3 |
| PRO | 168 | 8 | 114.3 |
| SER | 168 | 8 | 114.3 |
| THR | 168 | 8 | 114.3 |
| VAL | 168 | 8 | 114.3 |
| TRP | 168 | 8 | 114.3 |
| TYR | 168 | 8 | 114.3 |
| LEU | 140 | 8 | 137.1 |
| CYS | 168 | 8 | 114.3 |
| Sterile Deionized Water | | | 91.4 |
Table 2: Components of optimized V. natriegens cell-free reaction master mix for DNA template
| Cell-free Reaction Master Mix | |
| Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) 1x Reaction | Quantity (µL) 50x Reactions |
| Extract (%) | | 25 | 2.50 | 125.00 |
| Mg-glutamate | 100 | 3.5 | 0.35 | 17.50 |
| K-glutamate | 2000 | 80 | 0.40 | 20.00 |
| 4x Amino Acid Master Mix | 8.0 | 2 | 2.50 | 125.00 |
| 10x Energy Solution Master Mix | | | 1.00 | 50.00 |
| Plasmid DNA (ng/µL) | 1000 | 500 | 0.50 | 25.00 |
| 50% PEG-8000 (%) | 50 | 2 | 0.40 | 20.00 |
| T7 RNA Polymerase | | | 1.00 | 50.00 |
| RNase Inhibitor, Murine | | | 0.10 | 5.00 |
| Sterile Deionized Water | | | 1.25 | 62.50 |
| Reaction Volume (µL): | 10 | | | |