Refinement of OnePot PURE and Crude Ribosome Production for Reproducible Cell-free Protein Synthesis

We refined the OnePOT PURE system and crude ribosome preparation to improve reproducibility and accessibility, enabling more labs to build and troubleshoot robust cell-free systems for synthetic biology research. Experimental challenges include batch-to-batch variability, expression vector instability, and slow-growing strains, all of which hinder reproducibility and consistent performance in preparing in-house PURE systems. We established that careful control of strain growth, inoculation, and glycerol concentration significantly improves reproducibility and yields in OnePOT PURE and ribosome preparations across users and biological replicates.

We address the lack of a reproducible, accessible protocol for preparing PURE and ribosomes in house, helping labs overcome technical barriers to adopt self resistance for synthetic biology research. Our protocol improves reproducibility and flexibility by standardizing growth conditions and glycerol use and streamlines ribosome preparation using only ultracentrifugation, avoiding more complex or costly purification methods. To begin, thaw the frozen BL21 cell pellets on ice and add 50 milliliters of Ribosome Buffer A into a clean conical tube.

Using a pipette, take 100 microliters of Ribosome Buffer A from the conical tube and add it to a No-Weigh DTT vial. Aspirate gently until the DTT pellet is completely dissolved to create a 0.5 molar DTT stock solution. Then, pipette 100 microliters of the 0.5 molar DTT stock solution back into the 50 milliliter conical tube and place the tube on ice.

Using a vortex mixer, re-suspend each thawed cell pellet in Ribosome Buffer A with DTT at a ratio of two milliliters per one gram of cell pellet. Combine the resuspended samples into a conical tube and check that the total slurry volume is 10 milliliters. Now, divide the 10 milliliter slurry equally into two separate five-milliliter samples in labeled conical tubes.

Secure the conical tubes in a stand and lyse the cells using a cooled sonication probe in an ice water bath. Set the sonicator to 30%amplitude with a cycle of 20 seconds on and 20 seconds off for a total of 10 cycles. After sonication, top up each lysed sample to 25 milliliters using Ribosome Buffer A with DTT and briefly vortex to mix.

Transfer the 25-milliliter lysed samples into two pre-chilled S30 centrifugation tubes. Place the tubes in a centrifuge and spin at 30, 000 G for one hour at four degrees Celsius. Then, carefully recover up to 75%of the pellet-free supernatant from each S30 tube to prevent membrane contamination.

Transfer the recovered supernatant into two clean precooled S30 tubes. After centrifuging the tubes again for 30 minutes, recover up to 75%of the pellet-free supernatant and transfer it into two clean 50-milliliter comical tubes. Add Ribosome Buffer A with DTT to each tube until the volume reaches 25 milliliters.

Transfer the 25 milliliter samples into two clean, prechilled S100 ultracentrifuge tubes. Load the tubes into the ultracentrifuge and spin 100, 000 G for four hours at four degrees Celsius. After the final S100 centrifugation, discard the supernatant.

Use a pipette to remove any residual buffer while working quickly to prevent drying of the pellet. Then add 400 microliters of ice-cold Ribosome Buffer C with DTT into each S100 tube containing the ribosomal pellet. Incubate the tubes on ice overnight in a cold room to allow the ribosome pellet to soak in the buffer.

The following day, use a one-milliliter pipette and gentle swirling to fully re-suspend the clear halo ribosome pellet in buffer. Make sure only the clear halo is dissolved and avoid disturbing any yellow insoluble pellet. Then transfer the resuspended ribosome solution into two sterile two-milliliter microcentrifuge tubes.

Centrifuge the tubes at 20, 000 G for 10 minutes at four degrees Celsius to remove insoluble material. Carefully transfer the pellet-free supernatant into a clean microcentrifuge tube and keep it on ice. Thaw the necessary aliquots of PURE system proteins, ribosomes, energy solution, and matrix solution on ice.

Assemble a master mix for triplicate reactions by adding energy solution, PURE proteins, ribosomes, matrix solution, and nuclease-free water in the specified order. Set a pipette to 5.1 microliters and aspirate the master mix five to six times to ensure the solution is well mixed and homogeneous. Using the pipette set to 5.1 microliters, dispense the master mix into three separate wells of a 384-well optical plate, while avoiding bubbles.

Seal the 384-well plate using an adhesive plate seal. All four PURE reaction batches prepared by user 1 showed consistent GFP fluorescence kinetics, peaking around two hours, and were comparable to the GFP signal from the independently prepared batch by user 2.1, while no fluorescence was observed in the negative control lacking DNA. At the two-hour time point, the GFP fluorescence for all user-prepared PURE batches was similarly high, while the minus DNA control remained near baseline.

In ribosome preparations, GFP production over time varied slightly more across users with user 1.2 showing the highest activity, followed by users 2.1 and 3.1, while user 1.1 had the lowest among active batches. At 1.5 hours, user 1.2's ribosome preparation yielded the highest GFP fluorescence, while users 2.1 and 3.1 had similar but slightly lower values. User 1.1 had the lowest among active samples, and the minus DNA control remained near zero.

The overall variation between users 1.1 and 1.2 is around 30%GFP production in CFPS reactions decreased progressively with increasing glycerol concentrations above 0%with 6%showing the lowest expression.