This protocol simplifies and clarifies the methods for implementing self re-protein synthesis by non-experts. Improved access to these methods will help demarketize the platform and the broad set of applications that it enables. The main advantages of this technique are the speed, cost-effectiveness, and the ease of reaction set up compared to other self re-protein synthesis platforms.
Our platform can enable a number of applications including functional genomics, hythopertesting, biosensors, educational kits, and with minor modifications, metabolic engineering and genetic code expansion. While this technique only requires basic laboratory training, new users should plan to familiarize themselves with techniques like sonication for successful execution of the protocol. To begin this procedure prepare all media and culture E.coli cells as outlined in the text protocol.
Place a one liter centrifuge bottle into an ice water bath. Once the cultures OD600 reaches 3.0, pour the cell culture into the chilled bottle. Using a double beam balance, add water to a second one liter centrifuge bottle until it weighs the same as the first to create a balance for the centrifuge.
After pre-cooling the centrifuge to four degrees Celsius, centrifuge the bottles at 5, 000 g and at 10 degrees Celsius for 10 minutes to pellet the cells. After this, slowly pour off and dispose of the supernatant. Place the cell pellet on ice.
Using a sterile spatula scrape the cell pellet out of the centrifuge bottle and transfer it to a cold, 50 milliliter conical tube. Add 30 milliliters of cold S30 buffer supplemented with two millimolar DTT and resuspend the pellet by vortexing in short bursts with rest periods on ice until the pellet is fully resuspended with no chunks. After this, use another 50 millimeter conical tube filled with water as a balance to centrifuge the sample at 5, 000 g and 10 degrees Celsius for 10 minutes.
Pour off and dispose of the supernatant. Resuspend the pellet with 20 to 25 milliliters of cold S30 buffer. Centrifuge at 5, 000 g and at 10 degrees Celsius for 10 minutes.
Pour out and dispose of the supernatant. Then, add 30 milliliters of S30 buffer and resuspend the pellet using a vortex as previously described. Set out three pre-weighed, cold, 50 milliliter conical tubes.
Using a serological pipette filler with a sterile pipette, aliquot ten 10 milliliters of pellet suspension into each tube. Centrifuge all of the tubes using appropriate balances as needed at 5, 000 g and 10 degrees Celsius for 10 minutes. After this, pour out and dispose of the supernatants.
Carefully wipe the inside of each tube and cap with a clean tissue to remove the access buffer, while making sure to avoid touching the pellet. Using an analytical balance, reweigh the tubes and report the final pellet weight for each tube. To begin, add one milliliter of cold S30 buffer with 2 millimolar DTT per one gram of cell mass to each pellet.
Use a vortex to resuspend the pellets as previously described. Then transfer 1.4 milliliters of each resuspended pellet into separate 1.5 milliliter Microcentrifuge tubes. Place one tube into an ice water bath in a beaker and position the sonicator probe such that it does not touch the bottoms or sides of the tube.
Sonicate the tube with the amplitude set at 50%for 45 seconds, followed by 59 seconds of rest. Making sure to close and invert the tubes during the off periods. Immediately after sonication is complete, add 4.5 microliters of one molar DTT into the lysate.
Invert the tube several times to mix and then place it on ice. Repeat this process, sonicating and adding DTT for each tube of resuspended cells. Centrifuge the samples at 18, 000 g and four degrees Celsius for 10 minutes.
After this, pipette each supernatant into a new 1.5 milliliter Microcentrifuge tube, leaving some supernatant behind to ensure the pellet is not disturbed. Take the tubes of supernatant to the shaking platform of an incubator and incubate them at 37 degrees Celsius with shaking at 250 RPM for 60 minutes. This is the run-off reaction.
Then, centrifuge the samples at 10, 000 g and at four degrees Celsius for 10 minutes. Remove each supernatant without disturbing the pellet transferring them to new tubes. Create many 100 microliter aliquots of the extract for storage.
First, thaw solution A, solution B, the DNA template, the BL21DE3 extract, T7 RNA Polymerase, and an aliquot of molecular grade water on ice. Next, label the necessary amount of Microcentrifuge tubes needed for the CFPS reaction. Mix each reagent by pipetting or vortexing, then add the solution to the labeled reaction tube, making sure to not vortex the cell extract but instead invert the tube to mix.
Ensure that the final reaction is well mixed and combine into a single 15 microliter bead at the bottom of each tube. Incubate each reaction without shaking at 37 degrees Celsius for four hours, or at 30 degrees Celsius overnight. To begin, obtain a 96 well plate and load 48 microliters of 0.05 molar HEPES at pH 8 into each well needed for quantification.
Next, remove the reaction tubes from the incubator. Mix each reaction by pipetting up and down and transfer two microliters of each reaction into the wells containing HEPES. Mix each well by pipetting up and down.
After all reactions are loaded and mixed, load the plate into a fluorometer and measure the SFGFP end point fluorescents using an excitation wave length of 485 nanometers and an emission wavelength of 510 nanometers. Use a previously generated standard curve to determine the concentration of superfoldered GFP from the obtained fluorescents reading. In this study, a sonication based E.coli extract preparation is investigated.
Cell pellets and cell extract are stable at negative 80 degrees Celsius for at least a year. Additionally, the cell extract can undergo at least five freeze-thaw cycles without a significant loss of productivity. Some steps within this protocol can be varied without detriment to the overall productivity of the system.
Most notably, cells can be harvested within the range of 2.7 to 4.0 optical density at 600 nanometers without a significant effect on the cell extract productivity. However, template DNA quality is a source of batch-to-batch variability that impacts volumetric yields of the CFPS reaction. This is resolved by purifying the DNA via a Midi or Maxiprep followed by an additional DNA cleanup step.
The reaction vessel also impacts volumetric yields of the CFPS reaction. An increase in surface area to volume ratio, results in higher volumetric yields. To optimize volumetric yields of the CFPS reaction and to reduce cell extract batch-to-batch variability, a magnesium titration should be performed for each new extract preparation.
For cell extracts comprised of 30 milligrams per milliliter total protein, the optimal magnesium concentration and extract volume to minimize reagent usage and maximize volumetric yield is 10 millimolar and five microliters respectively. By varying the reaction scale, users can pursue methods ranging from high throughput screening to larger scale expressions of a target protein. This technique provides two key advantages, first it screens protein products more rapidly, and second, it synthesizes difficult to express proteins.
Examples include proteins that are cytotoxic or require oxidizing conditions for their function. As with any laboratory procedure, all reagents should be treated with caution. Additionally, centrifuges should always be balanced and ear protection should be used during sonication.
