Using this protocol, high-quality bacterial cell lysates can be produced using only broadly available common laboratory equipment, making cell-free gene expression easily accessible to most researchers. Combining program cellular autolysis with a freeze-thaw or freeze-dry cycle allows creating a practical, labor and cost effective approach for rapid production of bacterial lysates for cell-free gene expression. Begin by using an inoculation loop to streak the cells of an autolysis E.coli strain onto LB agar plates containing 50 micrograms per milliliter ampicillin, then incubate the plates at 37 degrees Celsius overnight.
The following day, using a pipette tip, pick a single colony from the agar plate and add it into a culture tube containing LB medium with ampicillin. Grow this starter culture at 37 degrees Celsius overnight. The next day, add 400 microliters of the starter culture into a one liter Erlenmeyer flask containing 400 milliliters of 2xYTPG medium supplemented with 50 micrograms per milliliter ampicillin.
Incubate the culture at 37 degrees Celsius with shaking at 300 RPM. Using a spectrophotometer and an optical cuvette with a one centimeter path length, periodically measure the culture's optical density at 600 nanometers. When the optical density exceeds one, begin diluting the culture five-fold before measurements to ensure that the measurements remain within the linear range of a typical laboratory spectrophotometer.
Once the optical density of the five-fold diluted culture reaches 0.3, remove the flask from the incubator and proceed to prepare the lysate. To prepare the lysate, harvest the cells by centrifugation at 1, 800 x g for 15 minutes at room temperature. Pour off the supernatant and remove the remaining liquid using a pipette.
Add 45 milliliters of cold S30A buffer to the pellet and resuspend the pellet by vortexing. Then, weigh an empty 50 milliliter centrifuge tube before transferring the cells into the tube. Centrifuge the cells again and discard the supernatant.
Aspirate any remaining supernatant using a pipette. Weigh the tube again and subtract the weight of the empty tube from the weight displayed to obtain the weight of the pellet. For every one milligram of cell pellet, add two microliters of cold S30A buffer supplemented with two millimolar dithiothreitol.
Then, resuspend the cells by vigorous vortexing. Next, freeze the cells by placing them in a negative 20 or negative 80 degrees Celsius freezer until the pellet is thoroughly frozen, then thaw the cells in a room temperature water bath and vortex vigorously for two to three minutes. Incubate the cells at 37 degrees Celsius for 45 minutes with shaking at 300 RPM, then clear the sample of heavy cellular debris by centrifuging in transparent centrifuge tubes at 30, 000 x g for 45 minutes at four degrees Celsius.
Carefully transfer the supernatant to a new tube with a pipette, taking care not to disturb the pellet. If the transferred supernatant is contaminated with material from the pellet, repeat the centrifugation. Transfer the supernatant to 1.5 milliliter centrifuge tubes.
And centrifuge once more at 21, 000 x g or the maximum speed of a tabletop centrifuge for five minutes. Aliquot the cleared autolysate into desired volumes and store at minus 80 degrees Celsius until use. For cell-free gene expression using the autolysate, mix eight microliters of autolysate and 8.9 microliters of pre-mix on ice.
Add DNA to a final concentration of eight nanomoles, any other necessary reagents and water to obtain a final volume of 20 microliters. Add the reaction to a 384-well microplate and use a plate reader to measure the fluorescence time course and endpoints. GFP expression using the autolysate with a dilution series of plasma DNA results in strong expression even with one nanomolar DNA.
Upon comparing the GFP expression between a commercially available lysate and the autolysate, the autolysate produced comparable levels of GFP. Variability between batches of lysate produced in two different labs by different researchers was within about two-fold. The three most critical variables are cell pellet to buffer ratio, transferring debris-free supernatant and using optimal PEG and magnesium concentration in the final reaction.
Once the extract has been prepared, a large arsenal of existing protocols developed for E.coli cell-free expression systems can be used, such as ClpXP-mediated degradation, or AHL-mediated quorum sensing.
