1. Media growth

1. Prepare 960 mL of CFAI media as described in Table 1 and adjust the pH to 7.2 using KOH.
2. Transfer culture media to a 2.5 L baffled flask and autoclave for 30 min at 121 °C.
3. Prepare a 40 mL sugar solution as described in Table 1. Filter-sterilize the solution into a separate autoclaved glass container.
   NOTE: The sugar solution can be stored in a 30 °C incubator until further use.
4. Allow the media to completely cool to below 40 °C after autoclaving.
5. Prior to inoculation of the CFAI media, add the sugar solution directly to the CFAI media.
6. To inoculate the media, swipe a loopful of colonies from a previously streaked E. coli BL21 Star (DE3) plate and insert directly into the media. Swirl the loop into the media but avoid touching the sides of the container. Ensure that the streak plate is fresh, with viable cells.
7. Place the flask with the inoculated media in a 30 °C incubator while shaking at 200 rpm. Allow the culture to grow overnight. If cells are inoculated in the evening, the culture will reach an approximate optical density, measured at 600 nm (OD₆₀₀), of 10 the subsequent morning. Inoculation and harvest times can be adjusted as needed.

2. Cell harvest

1. Prepare S30 buffer in advance and keep it cold. Prepare the S30 buffer according to Table 2, to a final pH of 8.2.
   NOTE: The S30 buffer can be prepared days prior to the cell harvest. If this is done, prepare without dithiothreitol and store at 4 °C. Add dithiothreitol just before use.
2. From this point forward, keep all solutions and materials on ice. Transfer 1 L of media into a 1 L centrifuge bottle and centrifuge at 5,000 x g between 4-10 °C for 10 min. Decant and dispose of the supernatant. Using a sterile spatula, transfer the pellet to a pre-chilled and previously weighed 50 mL conical tube.
3. Wash once with 30-40 mL of cold S30 buffer by resuspending the pellet via vortexing in 30 s bursts with rest periods on ice.
   NOTE: Due to a higher volume of pellet, splitting the cell pellet into two 50 mL conical tubes can be helpful for the wash step. Storing cells in smaller aliquots also provides flexibility for the downstream processing.
4. Centrifuge the cell resuspension at 5000 x g between 4-10 °C for 10 min.
5. Dispose of the supernatant and wipe any excess from the inside walls of the 50 mL conical tube using a clean tissue, avoid touching the pellet itself. Weigh and flash freeze the pellet in liquid nitrogen. Store at -80 °C until further use.
   ​NOTE: Pellets may not require flash-freezing if the user plans to continue with the extract preparation protocol.

3. Extract preparation

1. Combine the frozen pellet with 1 mL of S30 buffer for every 1 g of the cell pellet and allow to thaw on ice for approximately 30-60 min. Resuspend thawed pellet via vortexing in bursts of 30 s with rest periods on ice. Vortex until there are no visible clumps of cells remaining.
   NOTE: Smaller clumps can be resuspended by mixing using a pipette.
2. Transfer aliquots of 1.4 mL of cell resuspension into 1.5 mL microfuge tubes for cell lysis. Sonicate each tube with a frequency of 20 kHz and 50% amplitude for three bursts of 45 s with 59 s of rest per cycle surrounded by an ice bath. Invert the tube between cycles and immediately add 4.5 µL of 1 M dithiothreitol after the last sonication cycle.
   NOTE: Due to the heat released from sonication, it is extremely important to make sure all aliquots are kept on ice when not being sonicated. The ice bath should be constantly replenished or large enough to stay cool throughout the entire sonication process.
3. Centrifuge each tube at 18,000 x g and 4 °C for 10 min. Retrieve the supernatant and aliquot into 1.5 mL microfuge tubes in 600 µL aliquots. Flash freeze aliquots and store at -80 °C until further use. Care should be taken to pipette only the supernatant.

4. Cell-free protein synthesis

1. Thaw one aliquot of extract from the previous step to perform 15 µL of cell-free protein synthesis reactions in 1.5 mL microfuge tubes in quadruplicate.
2. Prepare each reaction by combining 240 ng of DNA (16 µg/mL final concentration), 2.20 µL of Solution A, 2.10 µL of Solution B, 5.0 µL of extract, and a varying volume of molecular-grade water to fill the reaction to 15 µL. This reaction can be scaled to higher volumes. See Table 3 for ratios.
   1. Prepare Solution A and B according to Table 4. Each solution can be prepared in batches of 100 µL to 1 mL, aliquoted, and stored at -80 °C until further use.
      NOTE: The DNA amount can vary depending on the protein of interest. In this case, the plasmid used, pJL1-sfGFP, has been optimized to perform at 16 µg/mL, or 597 µM.
3. Let the reactions run for at least 4 h at 37 °C.

5. Quantification of reporter protein, super folder green fluorescent protein (sfGFP)

1. Using a half area 96-well black polystyrene plate, combine 2 µL of each cell-free protein synthesis reaction product with 48 µL of 0.05 M HEPES buffer at pH 7.2. Three to four replicates of each reaction tube are recommended.
2. Quantify the fluorescence intensity of the sfGFP with an excitation wavelength of 485 nm and an emission wavelength of 528 nm.
3. For conversion of relative fluorescence units to volumetric yield (µg/mL) of sfGFP, establish a standard curve using purified pJL1-sfGFP.