The cell-free autoinduction method reduces researcher oversight and time needed to produce functional cell extracts. This simplifies previous in vitro protein synthesis protocols that were more time-consuming and intensive. This technique is less complicated and also high yielding, which will enable a larger audience to implement cell-free expression.
This method highlights the importance of energy metabolism during cell growth and within cell-free reactions. Begin by preparing 960 milliliters of cell-free autoinduction media. Then, sterilize the media in a 2.5-liter baffled flask by autoclaving for 30 minutes at 121 degrees Celsius.
Next, after preparing 40 milliliters of the sugar solution, filter-sterilize it into a separate autoclaved glass container. After autoclaving, when the culture media has cooled down to below 40 degrees Celsius, add the filter-sterilized sugar solution directly to the media. Next, inoculate the media by swiping a loop full of colonies from a previously streaked fresh E.coli BL21 star plate and inserting the loop directly into the media.
Swirl the loop into the media without touching the sides of the container. Then, place the inoculated media overnight in a 30 degree Celsius incubator with shaking at 200 RPM. The next day, harvest the cells by transferring one liter of the media into a one-liter centrifuge bottle and centrifuging at 5, 000 times G between four to 10 degrees Celsius for 10 minutes.
After discarding the supernatant, use a sterile spatula to transfer the pellet to a pre-chilled and previously weighed 50-milliliter conical tube. Next, wash the pellet once with 30 to 40 milliliters of cold S30 buffer by resuspending the pellet via vortexing in 30-second bursts with rest periods on ice. After the pellet is completely resuspended, centrifuge the cell resuspension, discard the supernatant, and using a clean tissue, wipe any excess supernatant from the inside walls of the 50-milliliter tube without touching the pellet.
Then, weigh the pellet before flash freezing it in liquid nitrogen and store it at minus 80 degrees Celsius until further use. To prepare the cell extract, add one milliliter of S30 buffer per gram of the frozen cell pellet and allow the pellet to thaw on ice for 30 to 60 minutes. Then, resuspend the thawed pellet via vortexing in bursts of 30 seconds with rest periods on ice until no visible cell clumps remain.
For cell lysis, transfer 1.4 milliliter aliquots of the cell suspension into 1.5-milliliter microfuge tubes. Then, sonicate the cell suspension in each tube with a frequency of 20 kilohertz and 50%amplitude for three bursts of 45 seconds with 59 seconds of rest per cycle in an ice bath Invert the tube between cycles, and after the last sonication cycle, immediately add 4.5 microliters of one-molar dithiothreitol. After sonicating the cell suspension in all tubes, centrifuge the tubes and collect the supernatant in 600-microliter aliquots into fresh 1.5-milliliter microfuge tubes.
Flash freeze and store the aliquots at minus 80 degrees Celsius until further use. To perform 15 microliters of cell-free protein synthesis reaction in 1.5-milliliter microfuge tubes in quadruplicate, thaw one aliquot of the cell extract. After preparing each 15-microliter reaction mixture, allow the reaction to run for at least four hours at 37 degrees Celsius.
To quantify the reporter protein, combine 48 microliters of 50-millimolar HEPES buffer at pH 7.2 with two microliters of each cell-free protein synthesis reaction product in a black polystyrene 96-well plate. Quantify the fluorescence intensity of the superfolder green fluorescent protein, or sfGFP, using an excitation wavelength of 485 and emission wavelength of 528 nanometers. Then, convert the relative fluorescence units to the volumetric yield of sfGFP by creating a standard curve using purified pJL1 sfGFP plasmid.
Shown here are cell-free autoinduction media pellets after cell harvest at different optical densities measured at 600 nanometers. The media grown to an optical density of 10 produced a higher amount of cell pellet in overall extract than the media grown to an optical density of 2.5. Further analysis of total protein concentration demonstrated no significant difference in overall protein between both extracts.
Even though the media was grown to different optical density levels, both extracts demonstrated similar results in cell-free reactions expressing sfGFP. Maintaining sterile conditions while preparing the media and sugar solution is important. This CFAI cell-extract method can be used for most applications of cell-free expression.
These range from biotechnology education to protein engineering, as well as point-of-care diagnostics. This method reduces the amount of time and technical skill required, improves reproducibility, and produces higher quantities of extract.
