OnePot PURE Cell-Free System

NOTE: This protocol describes the preparation of cell-free TX-TL system from recombinant components. For convenience, the work is separated into five parts. The first part describes preparation steps, which should be done before starting the protocol. The second part describes the preparation of the OnePot protein solution. The third part describes ribosome purifications, the fourth part details the preparation of the energy solution, and the last part provides a manual for setting up a PURE reaction. For convenience, the protocols are divided into days and summarized in daily schedules in Table 1. Following the schedule, the whole system can be prepared in 1 week by one person.

1. Preliminary work

1. Prepare the bacterial culture media and media supplements as described in Supplementary Table 1. Prepare and sterilize the materials required, including pipette tips, 96 deep-well plates.
2. Strain preparation
   1. Transform the expression strains indicated in Table 2 with the corresponding expression vectors using the heat shock method.
      1. Add purified plasmid to the chemically competent bacteria and incubate on ice for 20-30 min.
      2. Place the mixture at 42 °C for 30 s (heat shock) and then place it back on ice for 2 min.
      3. Pipette 20 µL of the bacteria directly onto agar plates containing ampicillin (AMP) and incubate at 37 °C overnight. Store the plates at 4 °C for up to 1 week.
      4. Inoculate 3 mL of LB media containing AMP with a single colony of bacteria from the agar plates. Incubate at 37 °C while shaking at 260 rpm overnight.
      5. Mix 250 µL of the culture with 250 µL of 50% (v/v) glycerol and store at -80 °C.
         NOTE: For faster preparation in the future, store the strains in a 96-well plate as glycerol stocks.
   2. Confirm all vector transformations by colony PCR and sequencing. Sequence the gene, promoter region, and ribosome binding site.
3. Expression test
   1. Inoculate 300 µL of LB media containing AMP with around 1 µL of the prepared glycerol stocks in a 1.3 mL deep-well plate. Seal the plate with a breathable membrane and then incubate at 37 °C while shaking at 260 rpm overnight.
      NOTE: All expressions are done separately at this point.
   2. Inoculate 300 µL of fresh LB media containing AMP with 1 µL of the overnight cultures. Incubate at 37 °C while shaking at 260 rpm overnight. After 2 h, induce the cells with 100 µM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grow for an additional 3 h.
   3. Mix 10 µL of the culture with 10 µL of 2x Laemmli buffer and heat to 95 °C for 10 min. Spin the samples for 1 min using a table centrifuge and load 10 µL of the supernatant on a PAGE gel. Run the gel in Tris/Glycine/SDS buffer at 200 V for 30 min. Rinse it well with deionized water. Cover the gel with a Coomassie protein stain and incubate for 1 h. Destain the gel in water if necessary (representative results for the expression test in Figure 1).
      NOTE: Use gradient (4%-15% or 4%-20%) PAGE gels to achieve a good separation.
4. IMAC Sepharose resin restoration and cleaning
   1. Column preparation.
      1. Mix the Sepharose resin well by vortexing.
      2. Pipette the required amount of resin into an empty gravity flow column.
         NOTE: The amount of resin required varies between His-ribosome purification and protein purification and is specified in the respective sections.
      3. Wash the resin with 30 mL of deionized water.
      4. Proceed with column re-charge as specified in section 1.4.4.
         NOTE: Always let all the liquid pass through the column before continuing with the next step. However, make sure that the column never runs dry. Whenever running any liquid through the column, ensure to stop the flow or continue to the next step as soon as the liquid reaches the resin.
   2. Restoration.
      1. Wash the column with 30 mL of deionized water.
      2. Apply 10 mL of a 0.2 M EDTA and 0.5 M NaCl solution.
      3. Add 30 mL of a 0.5 M NaCl solution.
      4. Wash the column with 50 mL of deionized water.
      5. Store in 20% (v/v) ethanol at 4 °C or continue with the next step.
   3. Cleaning.
      CAUTION: Wear protective equipment.
      1. Wash the column with 30 mL of 0.5 M NaOH.
      2. Wash the column with 30 mL of deionized water.
      3. Wash the column with 30 mL of 0.1 M acetic acid.
      4. Wash the column with 30 mL of deionized water.
      5. Wash the column with 30 mL of 70% (v/v) ethanol.
      6. Wash the column with 50 mL of deionized water.
      7. Store in 20% (v/v) ethanol at 4 °C or continue with the next step.
   4. Re-charging.
      1. Add 10 mL of 0.1 M nickel sulfate solution to the column.
         CAUTION: Nickel sulfate is toxic. Nickel sulfate waste needs to be discarded with the precautions indicated by the supplier.
      2. Wash the column with 50 mL of deionized water.
      3. Store in 20% ((v/v)) ethanol at 4 °C or continue with the column equilibration.
         ​NOTE: If the column is stored in ethanol between steps, make sure to remove all traces of ethanol by washing the column with water.

2. OnePot protein solution expression and purification

NOTE: The protocol consists of three parts divided into days (Figure 2). An ideal preparation procedure produces 1.5 mL of 13.5 mg/mL OnePot protein solution, which corresponds to more than one thousand 10 µL PURE reactions. However, the amount and the ideal concentration of the solution will vary from batch to batch. Experienced users can perform multiple OnePot PURE preparations at a time.

Day 1:

1. Prepare bacterial culture media and media supplements as described in Supplementary Table 1.
2. Prepare and sterilize the required materials, including pipette tips, two 96 deep-well plates, and one 1 L baffled Erlenmeyer flask.
3. Prepare buffers and supplements as described in Supplementary Table 2. Filter sterilize all buffers using bottle top filters (0.45 µm) and store them at 4 °C. Supplement all the buffers with 1 mM TCEP right before use, unless indicated otherwise.
4. Use 2 mL of sepharose resin for the OnePot protein purification. Prepare the column as described in section 1.4.
5. To prepare the starter cultures, combine 20 mL of LB media with 20 µL of AMP. In a sterile 96, 1.3 mL deep-well plate, add 300 µL of the media into 35 wells. Inoculate each of them with its respective strain, except elongation factor thermo unstable (EF-Tu), and seal the plate with a breathable membrane.
   NOTE: Inoculate the plate using a 96-well replicator (see Table of Materials). The well volume of the deep-well plate and the volume of the starter culture are essential. Larger media volumes or smaller well volumes will lead to a different bacterial density due to aeration inconsistencies.
6. For the EF-Tu culture, inoculate 3 mL of LB media in a 14 mL culture tube with a snap cap. A single 3 mL of culture for EF-Tu is sufficient for one OnePot expression culture.
7. Incubate at 37 °C while shaking at 260 rpm overnight.

Day 2:
NOTE: Perform all the steps at room temperature unless indicated otherwise.

8. Transfer 500 mL of LB media and 500 µL of AMP into the sterile baffled flask.
9. Inoculate the OnePot PURE culture with 1675 µL of the EF-Tu culture and 55 µL of each of the cultures from the deep-well plate (Table 2).
   NOTE: During this step, the overall protein composition can be adjusted by tuning the inoculation ratios. Make sure that the overall inoculation volume remains constant at 3.6 mL.
   OPTIONAL: To confirm that all strains have grown overnight, measure the optical density of the overnight cultures at 600 nM (OD₆₀₀) in a 96-well plate using a plate-reader. Use a dilution of 10x for the optical density measurement.
10. Incubate the culture for 2  h at 37 °C with a shaking of 260 rpm, or until the OD₆₀₀ of the culture reaches 0.2-0.3.
11. Induce the culture with 500 µL of 0.1 mM IPTG and grow for an additional 3 h.
12. Harvest the cells by centrifugation at 4 °C and 3220 x g for 10 min and store the cell pellet at -80 °C until further use.
    NOTE: To optimize the timing, prepare the energy solution described in section 4 during the incubation times on day 2 (Table 1).

Day 3:

13. Measure the amounts of buffers needed for the purification described in the steps below and add TCEP to all of them as indicated in Supplementary Table 2. Store the remaining buffers without TCEP at 4 °C for future purifications.
14. Equilibrate the charged column (section 2.4) with 30 mL of buffer A. After 25 mL of buffer A have passed through, close the column from the bottom. In parallel, continue with steps 2.15-2.17.
15. Thaw the cells and use a serological pipette to resuspend the cell pellet in 7.5 mL of buffer A.
16. Lyse the cells using a 130-watt probe sonicator (see Table of Materials, probe tip diameter: 6 mm) with the following parameters: 4 x 20 s pulse on, 20 s pulse off, 70% amplitude. If sonication is successful, the solution will turn darker (Figure 2).
    NOTE: Make sure to keep the cells on ice during sonication. Place the probe deep enough into the solution without touching the tube. If a large amount of foam is generated, the energy transfer will be damped. In that case, let the foam settle, lower the probe deeper into the solution, and extend the sonication time.
17. Remove the cell debris by centrifugation at 21130 x g for 20 min at 4 °C immediately after sonication. Keep the lysate on ice.
18. Add the supernatant to the equilibrated column. Close the column from the top and make sure there is no leakage. Incubate the column for 3  h at 4 °C under rotation using a tube rotator.
19. Elute unbound components from the column and wash with 25 mL of buffer A.
20. Wash the column with 25 mL of 25 mM imidazole buffer (23.95 mL of buffer A and 1.25 mL of buffer B).
21. Elute the proteins with 5 mL of 450 mM imidazole buffer (0.5 mL of buffer A and 4.5 mL of buffer B). Keep the eluted proteins on ice at all times.
22. Dilute the eluate with 25 mL of HT buffer, keep the mixture on ice. Add 15 mL to a 15 mL centrifugal filter and concentrate to a volume of 1.5 mL. Add the remaining 15 mL to the filter with the concentrated solution and concentrate to 1.5 mL once more.
23. Add 10 mL of HT buffer to the concentrated sample and concentrate to 1 mL. Add an equal amount of stock buffer B and store at -80 °C until further use.
    NOTE: One round of exchange/concentration takes about 60 min spinning at 3220 x g at 4 °C.
24. During the buffer exchange, restore the column as specified in section 1.4.

Day 4:

25. Measure the protein concentration using the Bradford assay as described by the supplier. Concentrate the sample with a 0.5 mL of 3 kDa cutoff centrifugal filter to 20 mg/mL.
    NOTE: Dilute the protein solution 25-fold or 50-fold before the concentration measurements to avoid oversaturating the Bradford assay.
26. To establish the ideal protein concentration, perform an expression test at this stage (section 5.2) with different concentrations of the protein solution. To perform the titration, keep the total volume of the solution constant and pipette the OnePot protein solution, including stock buffer B, at five different ratios (Supplementary Table 7).
27. Verify the OnePot PURE protein composition using SDS-PAGE (Figure 3A). Dilute 2.5 µL of the sample with 7.5 µL of water, mix with 10 µL of 2x Laemmli buffer and then load 5 µL and 2.5 µL of the samples to the gel. Run the SDS-PAGE as specified in section 1.3.3.
28. Aliquot the protein solution into 50 µL aliquots after verifying the expression and adjusting the concentration. Store the OnePot PURE protein solution at -80 °C until further use.
    NOTE: If a protein component is suspected not to be present, or is present in a lower-than-expected concentration in the OnePot PURE, perform the following steps.
29. Check whether the overnight culture of the respective strain has grown at a comparable rate to the other cultures by performing optical density measurements (OD₆₀₀) of all cultures.
30. Perform an additional expression test of the specific strain to verify the expression of the suspect protein.

3. Ribosome solution

NOTE: Two different ribosome purification strategies are introduced, one for hexahistidine-tagged and one for non-tagged ribosomes. The major advantage of the purification method using His-purification on a standard affinity Ni-NTA gravity flow column is that the purification is easy, fast, and does not require additional laboratory equipment, such as a FPLC system and an ultracentrifuge. However, the protein production capacity in OnePot PURE reactions is around one-third compared to tag-free ribosomes. Therefore, choose the method for ribosome production based on whether a high yield is important for the given application.

1. His-tagged ribosome purification
   NOTE: This protocol utilizes the E. coli RB1 strain, a gift from Professor Wang (Columbia University, USA)¹⁸. This strain has a genomic insertion of a hexahistidine tag on the C terminus of 50S ribosomal protein (L7/L12), allowing for purification using a Ni-NTA gravity-flow column. The usual yield is around 0.5 mL of 3.45 µM ribosomes, which is sufficient for more than five hundred 10 µL PURE reactions.

Day 1:

1. Prepare bacterial culture media and media supplements as described in Supplementary Table 1.
2. Prepare and sterilize the required materials, including pipette tips, one 5 L Erlenmeyer flask, and one 100 mL Erlenmeyer flask.
3. Prepare buffers and supplements as described in Supplementary Table 2. Filter sterilize all the buffers using bottle top filters (0.45 µm) and store them at 4 °C.

Day 2:

4. Pipette 5 mL of resin to a column and prepare the column as specified in section 1.4.
   NOTE: Due to the higher volume of the resin, the restoration and purification take significantly longer. Use a different column for ribosome purification to avoid cross-contamination and thoroughly clean it before the purification.
5. Prepare an overnight culture of E. coli RB1 strain by inoculating 35 mL of LB media in a 100 mL Erlenmeyer flask. Incubate at 37 °C while shaking at 260 rpm.

Day 3:
NOTE: Perform all the steps at room temperature unless indicated otherwise.

6. Add 2 L of LB media into a 5 L sterile flask, inoculate with 12 mL of the overnight culture, and then incubate for 3-4 h at 37 °C while shaking at 260 rpm.
   NOTE: Alternatively, perform bacterial culturing in 4 x 500 mL of cultures in 1 L baffled flasks.
7. Pellet the cells by centrifugation for 10 min at 3220 x g and 4 °C. Store at -80 °C until further use.

Day 4:

8. Equilibrate the column prepared in step 3.1.4. with 30 mL of lysis buffer.
9. Resuspend the pellet in 20 mL of lysis buffer using a serological pipette.
10. Lyse the cells with a 130-watt probe sonicator (see Table of Materials, probe tip diameter: 6 mm) on ice with the following parameters: 11 x 20 s pulse on; 20 s pulse off, 70% amplitude (see step 2.16 for procedure details).
11. Immediately after sonication, remove the cell debris by centrifugation for 20 min at 21130 x g at 4 °C. Keep the lysate on ice.
12. Load the supernatant to the columns and let it pass through.
13. Wash the column with the following mixtures of lysis and elution buffers.
    1. Wash 0: use 30 mL of lysis buffer.
    2. Wash 1: use 30 mL of 5 mM imidazole (29 mL of lysis buffer, 1 mL of elution buffer).
    3. Wash 2: use 60 mL of 25 mM imidazole (50 mL of lysis buffer, 10 mL of elution buffer).
    4. Wash 3: use 30 mL of 40 mM imidazole (22 mL of lysis buffer, 8 mL of elution buffer).
    5. Wash 4: use 30 mL of 60 mM imidazole (18 mL of lysis buffer, 12 mL of elution buffer).
14. Elute the ribosomes with 7.5 mL of the elution buffer. Keep the eluted proteins on ice at all times.
15. Add 22 µL of pure β-mercaptoethanol to 45 mL of ribosome buffer.
    CAUTION: β-mercaptoethanol is toxic. Take safety precautions and work in a fume hood.
16. Add the eluate to a 15 mL centrifugal filter and concentrate to 1 mL.
17. Add 15 mL of ribosome buffer to the concentrated sample and concentrate again to 1 mL.
    NOTE: Repeat the previous step twice.
18. Store at -80 °C until further use.
    ​NOTE: One round of exchange/concentration takes about 60 min of centrifugation at 3220 x g at 4 °C.
19. During the buffer exchange, restore the column as specified in section 1.4.

Day 5:

21. Determine the ribosome concentration by measuring the absorbance at 260 nM of a sample diluted 1:100 in ribosome buffer. An absorbance value of 10 of the diluted solution corresponds to 23 µM of undiluted solution as previously described¹⁶.
22. Implement a final stock concentration of 3.45 µM. To adjust the concentration, dilute the ribosomes with ribosome buffer or concentrate them further by centrifugation at 14000 x g in a 3 kDa 0.5 mL centrifugal filter at 4 °C.
    NOTE: To achieve optimal system expression, perform a ribosome concentration titration (section 5.2, Supplementary Table 7).
23. Verify the ribosome composition using SDS-PAGE (Figure 3A) as specified in section 1.3.3. Dilute 2.5 µL of the sample with 7.5 µL of water, mix with 10 µL of 2x Laemmli buffer, and then load 5 µL and 2.5 µL of the samples onto the gel.

2. Tag-free ribosome purification
   NOTE: Tag-free ribosome purification is performed using a FPLC system (Table of Materials) and is based on hydrophobic interaction chromatography using 2 x 5 mL Butyl columns (Table of Materials). Although ribosomes may be purified from any strain, using the E. coli A19 (E. coli Genetic Resources at Yale CGSC) strain is advantageous due to its RNase I deletion²². Perform the purification at 4 °C in either a cold room or a cooling cabinet. The usual yield is around 0.5 mL of 10 µM ribosomes, which corresponds to more than five hundred 10 µL PURE reactions.

Day 1:

1. Prepare bacterial culture media and media supplements as described in Supplementary Table 1.
2. Prepare and sterilize the required materials, including pipette tips, 5 L Erlenmeyer flask, and 100 mL Erlenmeyer flask.
3. Prepare buffers and supplements as described in Supplementary Table 2. Filter sterilize all the buffers using bottle top filters (0.45 µm) and store them at 4 °C.

Day 2:

4. To prepare an overnight culture of the E. coli A19 strain, inoculate 35 mL of LB media in a 100 mL Erlenmeyer flask. Incubate at 37 °C while shaking at 260 rpm.

Day 3:

5. Transfer 2 L of LB media into the 5 L sterile baffled flask, inoculate with 30 mL of the overnight culture, and then incubate for 3-4  h at 37 °C while shaking at 200 rpm.
6. Pellet the cells by centrifugation at 4000 x g for 15 min at 4 °C. Resuspend the pellet in 25 mL of suspension buffer and store at -80 °C until further use.

Day 4:

7. Perform steps 3.2.8-3.2.12 in parallel with steps 3.2.13-3.2.19.
8. Thaw and lyse the cells using a 130-watt probe sonicator (see Table of Materials and probe tip diameter: 6 mm) on ice with the following parameters: 12 x 20 s pulse on; 20 s pulse off, 70% amplitude (see step 2.16 procedure details).
9. Immediately remove the cell debris by centrifugation at 20000 x g for 20 min at 4 °C.
10. Aspirate the supernatant and measure the volume. Add an equal volume of suspension buffer (high salt) to adjust the final concentration of ammonium sulfate to 1.5 M and mix well.
11. Remove the precipitate by centrifugation at 20000 x g for 20 min at 4 °C.
12. Filter the supernatant using a 0.45 µm polyethersulfone membrane syringe filter before FPLC purification and collect the filtrate in a 100 mL glass bottle. Keep the supernatant at 4 °C at all times.
13. Set up the FPLC system for hydrophobic-interaction chromatography purification using a double Butyl column (2 x 5 mL) as follows. For this setup, one column volume (CV) refers to a volume of 10 mL.
14. Three inlets will be needed: two as buffer lines and one as the sample line. Due to the default settings of the purifier, it is convenient to choose lines A1 and B1 for buffer C and buffer D, respectively, and line A2 as the sample line. Apply a default flow rate of 4 mL/min, except for pump washes (10 mL/min) or unless indicated otherwise.
    NOTE: As TCEP is a costly reagent, add the corresponding amount to buffers C and D only after the equilibration step.
15. Perform a system pump wash in 20% ((v/v)) ethanol to clean the system and remove potential contamination from previous purifications. Manually set a flow rate of 0.2 mL/min and mount the column. Stop the flow.
16. Execute a system pump wash with water. Wash the column with 3 CV of water.
17. Equilibration: place inlets A1 and A2 in buffer C and inlet B1 in buffer D without TCEP. Execute a pump wash and equilibrate the column with 4 CV of buffer C.
18. Add TCEP to buffers C and D.
19. Prepare 15 mL tubes or clear round fraction collector tubes to the fraction collector to collect 4-5 mL elution fractions.
20. Loading: Place the inlet A2 into the bottle with the filtered sample. Load approximately 90% of the sample volume onto the column. Dilute the sample with 20 mL of TCEP-containing buffer C, and load 10 mL of the sample onto the column. Repeat the dilution step at least twice and load as much sample onto the column as possible. It is critical to ensure that no air is sucked into the machine.
21. Washing step 1: wash with 3 CV of buffer C to remove the unbound components.
22. Washing step 2: wash with 5 CV of 80% buffer C and 20% buffer D.
23. Elution: elute the product by applying 50% of buffer C and 50% of buffer D, with a total elution volume of 5 CV. Collect this fraction in the collector tubes.
24. Washing step 3: Elute all strongly interacting contaminants using 100% buffer D with a total volume of 5 CV.
25. Analyze the absorption spectrum of the sample fraction at 260 or 280 nM (Figure 4). The first peak shows the non-absorbed proteins eluted during loading and the first washing step; the second peak shows contaminants that have been eluted during the second washing step. The third peak monitors the final product, and the last peak shows the strongly interacting contaminants. Pool all sample fractions corresponding to the third peak for further processing. Keep the eluted proteins on ice at all times.
26. Gently overlay the recovered fraction onto 15 mL of the cushion buffer in four polycarbonate ultracentrifugation tubes. Add a maximum of 15 mL of the sample to 15 mL of the cushion buffer. Make sure to balance the weight of the tube well. Pellet the ribosomes by ultracentrifugation at 100000 x g at 4 °C for 16 h.
    NOTE: Ensure that no cracks are present in the ultracentrifugation tubes.
27. Clean and reset the column as follows. A flow rate of 5 mL/min works well. Place all the inlets into the water and execute a pump wash. Wash the column with 2 CV of water.
    1. Place the inlet into a 0.5 M NaOH solution, perform a pump wash, and subsequently wash the column with 3 CV of NaOH.
    2. Place the inlet into water, perform a pump wash, and then wash the column in 2 CV of water.
    3. Place the inlet to a 0.1 M acetic acid solution, perform a pump wash, and subsequently wash the column with 3 CV of acetic acid solution.
    4. Pump wash and wash the column with 2 CV of water.
    5. Place all inlets into 20% ((v/v)) ethanol, execute a pump wash step, and store the column in 20% ((v/v)) ethanol by washing it with 3 CV of a 20% ((v/v)) ethanol solution.
       NOTE: Ensure that the system never runs dry or sucks in air. Never apply buffer directly to ethanol, or ethanol to buffer. Always add a water washing step in between, as otherwise there is a risk of precipitates clogging the column. Make sure to add enough sample collection tubes.

Day 5:

28. Discard the supernatant and carefully, without disturbing the translucent pellet, wash each pellet with 0.5 mL of ice-cold ribosome buffer. Repeat this step twice.
29. Resuspend each of the clear pellets in 100 µL of ribosome buffer on ice using a magnetic stir bar (3 mM diameter, 10 mM length) on a magnetic stirrer using the lowest possible speed. Collect the resuspended ribosomes and wash the tubes with an additional 50 µL of ribosome buffer.
    NOTE: The translucent pellet is difficult to see. Therefore, carefully wash the pellet from the sides of the tube.
30. Determine the ribosome concentration by measuring the absorbance at 260 nM of the sample diluted at a ratio of 1:100 in ribosome buffer. An absorbance of 10 of the diluted solution corresponds to 23 µM of undiluted solution as previously described¹⁶.
31. Implement a final stock concentration of 10 µM. To adjust the concentration, dilute the ribosomes with ribosome buffer or concentrate them further by centrifugation at 14000 x g in a 3 kDa centrifugal filter at 4 °C.
    NOTE: To achieve optimal system expression, perform ribosome titration (section 5.2, Supplementary Table 7).
32. Verify the ribosome composition with SDS-PAGE (Figure 3A) as specified in section 1.3.3. Dilute 2.5 µL of the sample with 7.5 µL of water, mix with 10 µL of 2x Laemmli buffer, and then load 5 µL and 2.5 µL of the samples to the gel.

4. Energy solution

NOTE: The composition for the 2.5x energy solution introduced here is an example of a solution that worked well for a standard TX-TL reaction. To optimize the timing, prepare the energy solution during day 2. The preparation of the amino acid solution is explained in detail, followed by the final preparation procedure.

1. Amino acid solution
   NOTE: Prepare the amino acid solution in bulk. Preparing the amount of amino acid stock solutions required for a final volume of at least 2000 µL will reduce the weighing error for the otherwise very small amounts. The overall concentration of the amino acid solution is limited by the solubility of the amino acids and the respective stock solution concentrations. For the standard PURE system, prepare a solution with a final concentration of 3.25 mM. Use the amino acid solution calculation table (Supplementary Table 3) as a template. Use cysteine in the salt form to ensure sufficient solubility. Avoid using KOH-based amino acid preparation methods. It is possible to directly weigh the exact amounts of amino acids into the final amino acid solution without preparing stock solution for all the amino acids. However, this is more challenging and less precise.
   1. Prepare stock solutions for each amino acid as described in Supplementary Table 3, except for Tyrosine.
      NOTE: Due to the different solubilities of the amino acids in water, the respective suggested concentrations of the stock solution differ.
   2. Minimal mass [mg] provides the approximate minimum mass required to obtain a sufficient amount of stock solution for the target overall volume, as a reference.
      NOTE: The minimal mass is calculated with a surplus of 10%.
   3. For an easier preparation of the solutions, do not weigh the exact amount of amino acid, but instead, for the mass at hand, adjust the amount of water to achieve the desired concentration. Calculate the amount of deionized water (Water to add [µL]) needed, based on the actual mass filled in (light yellow cells) and the desired concentration using the spreadsheet in Supplementary Table 3.
   4. Solubilize the amino acid stock solutions by vortexing until all precipitate has dissolved. The individual amino acid stock solutions can be stored at -20 °C for several weeks.
      NOTE: Some amino acids are difficult to dissolve in water; the process may take some time.
   5. Weigh the exact amount of tyrosine required to obtain a final concentration of 3.25 mM directly into the tube for the amino acid solution.
      NOTE: Tyrosine is very difficult to dissolve in water. Add it directly instead of preparing a stock solution.
   6. Add the corresponding amounts of amino acid stock solutions and water as indicated in the Final volume to add [µL] column (light blue cells) and vortex the solution well. Store the completed amino acid solution at -80 °C until further use.
2. Preparation of the energy solution
   NOTE: In total, the 2.5x energy solution contains 0.75 mM of each amino acid, 29.5 mM of magnesium acetate, 250 mM of potassium glutamate, 5 mM of ATP and GTP each, 2.5 mM of CTP, UTP, and TCEP, respectively, 8.75 mg/mL of tRNA from E. coli MRE 600, 50 mM of creatine phosphate, 0.05 mM of folinic acid, 5 mM of spermidine, and 125 mM of HEPES. First-time users prepare the energy solution in small batches of 200 µL. Store the individual solutions prepared according to Supplementary Table 4 at -20 °C or -80 °C for later use.
   1. Thaw all aqueous solutions mentioned in the Supplementary Table 5 on ice.
   2. Meanwhile, prepare the stock solutions for the remaining components listed in Supplementary Table 4. Keep all the solutions on ice after preparation.
      NOTE: Add 500 µL of RNase and DNase-free water directly to the vial to dissolve the lyophilized tRNAs. Mix well by gentle vortexing; limit pipetting to avoid introducing RNases.
   3. Add the calculated volumes (Supplementary Table 5) of stock solutions and water and mix well using a vortex. Keep the solution on ice at all times.
   4. Measure the pH of the solution by pipetting 1 µL onto a pH strip, to ensure that the pH of the solution is neutral.
   5. Aliquot the energy solution at 50-100 µL per tube on ice and store at -80 °C until further use. While aliquoting, vortex the main stock frequently to prevent the components from precipitating.
      ​NOTE: Optionally, conduct an activity assay of the newly made energy solution against commercial energy solutions, e.g. Solution A in PURExpress. If a significantly lower performance of the system with the energy solution is observed, optimizing the ion concentrations, especially magnesium ions, by titration (5-20 mM) may be advantageous.

5. OnePot PURE reaction

1. DNA template
   NOTE: Proteins encoded downstream of the T7 promoter can be expressed in PURE from either linear or circular DNA. By generating a linear DNA template using extension PCR, tedious cloning steps can be omitted. The linear templates for this study were generated by PCR as described below, using a high-fidelity DNA polymerase (Table of Materials). Primer sequences, melting temperatures, and the thermocycler settings used in this study are specified in Supplementary Table 6. The preparation of the DNA template is not included in the daily schedule.
   1. Set up a PCR reaction as recommended by the polymerase supplier.
      NOTE: Optimized parameters for a high-fidelity DNA polymerase (Table of Materials) are given in Supplementary Table 6.
   2. Amplify the target gene (e.g., eGFP) as a linear template from a plasmid or genome using gene-specific primers (500 nM) (for the parameters, see Supplementary Table 6).
   3. The amplification generates short extensions to provide annealing sequences for the following extension PCR steps.
   4. Check the amplicon on an agarose gel for correct size and purity.
   5. Use the amplified DNA as a template for the subsequent extension steps. Set up a reaction of at least 50 µL.
   6. Run 10 PCR amplification cycles with the extension primers (2.5 nM). After completing the amplification cycles, immediately add the final primers (500 nM) to the same reaction and run 30 cycles to amplify the extended PCR product. Find the melting temperatures and primer sequences in Supplementary Table 6.
   7. Purify the DNA fragments using a DNA purification kit and elute the DNA in nuclease-free water instead of EDTA containing elution buffer.
   8. Check the linear template on an agarose gel for correct size and purity.
   9. Measure the DNA concentration in ng/µL using an UV-Vis spectrophotometer.
2. Setting up the PURE reaction
   NOTE: The final reaction composition is 1x energy solution, tag-free ribosomes or His-tag ribosomes, OnePot PURE proteins, and DNA template. The reaction volume ratio comprises 40% energy solution, 30% protein and ribosome solution, and 30% DNA and water. Typical reaction volumes vary between 5 µL and 25 µL. Quantify the expression of a fluorescent protein continuously on a plate-reader. Use a Green Lys in vitro Translation Labeling System, which incorporates fluorescently labeled Lysine residue into newly synthesized proteins, to verify the expression of non-fluorescent proteins on a SDS-PAGE gel. An example reaction template is given in Supplementary Table 7 to help establish a PURE cell-free expression reaction. Cells in yellow indicate user-input values, and cells in orange indicate additional reagents to be optionally added to the reaction. Keep the volume ratios of the components precise to ensure the correct ion balance. For instance, to achieve a higher protein concentration, increase the OnePot protein solution concentration; however, do not increase the volume of protein solution added to the reaction.
   1. Fill in the concentration [ng/µL] and length [base pairs] of the DNA in the corresponding yellow cells in the spreadsheet. Use 2-10 nM of DNA for the reaction.
   2. Fill in the desired total reaction volume in µL.
   3. Remove the required reagents from the freezer and thaw them on ice.
      NOTE: Refreezing of the components is possible without a decrease in functionality. However, minimize the number of freeze-thaw cycles and the time samples are stored on ice as much as possible.
   4. Pipette the calculated amounts of water, DNA, and energy solution to one side of the PCR tube or one corner of a well on the 384-well plate. Add the required amount of any additional reagent on the same side. Minimize the number of samples per experiment to avoid sample evaporation and experimental start time bias.
      NOTE: It is crucial to keep the energy component physically separated from the protein components to avoid premature consumption of the energy sources and lower yields.
   5. Pipette the calculated amounts of protein and ribosome solution to the other side of a PCR tube or the opposite corner of the 384-well plate.
      NOTE: Use master mixes whenever possible to reduce the impact of pipetting errors. After initial testing, the ribosome and protein solutions can be mixed and stored as one solution.
   6. Spin for a short time (30 s) to merge the reaction components. To prevent evaporation during plate-reader experiments, add 35 µL of liquid wax and seal the plate with a transparent sealant (see Table of Materials).
   7. Incubate for a minimum of 3  h at 37 °C.
   8. For readout on a plate-reader, measure the fluorescence intensity at the required wavelength every 2 min (representative results are shown in Figure 3B).
   9. Perform the following steps for Green Lys labeled samples.
   10. After the cell-free expression, incubate the sample with 0.16 µg/µL of RNase A for 30 min at 37 °C to remove the fluorescent background of the Green Lys labeling kit.
       NOTE: Use RNase A, as other types of RNases do not remove the background sufficiently well.
   11. Visualize the protein expression by running SDS-PAGE as specified in section 1.3.3. Wash the unstained gel gently in deionized water, and image it on a fluorescent imager using an excitation wavelength of 488 nm.
   12. Subsequently, stain the gel using conventional Coomassie staining methods. For the suitable parameters see section 1.3.3.
       NOTE: Perform a titration of the protein solution with the recommended ribosome concentration and, if required, titrate ribosomes with the optimal OnePot protein concentration afterward. Use the commercial PURExpress ΔRibosome kit as a positive control. Solution A, Factor Mix, and the ribosome solution correspond to the prepared energy, the OnePot protein solution, and the purified ribosomes, respectively.