The protocol is very significant because it enables fast, easy, and direct use of linear DNA templates in cell-free protein synthesis systems, even from native E.coli parameters. The main advantages are the time amount saved by avoiding cloning, chemical modifications of linear DNA ends and protective supplements like gamma or chi DNA. As self-reporting synthesis extracts are being developed for different organisms of interest, our method presents a way of speeding up prototyping in those extracts by using linear DNA.
To begin, streak strain BL21 Rosetta-2 delta recBCD from minus 80 degrees Celsius glycerol stock onto a 2x YTP agar plate that contains 10 micrograms per milliliter chloramphenicol and incubate overnight at 37 degrees Celsius. Then, inoculate a single colony from the agar plate into a little more than 40 milliliters of 2x YTP supplemented with 10 micrograms per milliliter chloramphenicol and incubate overnight at 37 degree Celsius with 200 RPM shaking. Next, make a subculture by diluting the overnight culture 100 times into four liters of fresh 2x YTP media containing 10 micrograms per milliliter chloramphenicol.
Divide the four liters of the media into four separate flasks. Incubate at 37 degrees Celsius, 200 RPM, for about three to four hours of growth to reach an optical density of 1.5 to 2.0. Put the culture on ice, spin down the cells at 5, 000 G for 12 minutes at four degree Celsius, and then discard the supernatant by decanting.
Then, resuspend the cell pellets in 800 milliliters of chilled S30A with DTT. Again, spin down the cells at 5, 000 G for 12 minutes at four degree Celsius as demonstrated previously and discard the supernatant by decanting. After resuspending cell pellets in 160 milliliters of chilled S30A with DTT, transfer the cells to chilled and pre-weighed 50-milliliter tubes.
Spin down the cells at 2000 G for eight minutes at four degree Celsius and then discard the supernatant by decanting. Again, spin down the cells at 2000 G for four minutes at four degrees Celsius, and then remove the remaining supernatant carefully by using a pipette. Then, remeasure the weight of the tube to calculate the weight of the cell pellet and keep the cell pellets at minus 80 degrees Celsius.
After taking the cell pellets from minus 80 degrees Celsius, thaw them on ice for one to two hours and resuspend the cell pellets in 0.9 milliliters of S30A with DTT buffer per gram of the cell pellet's weight. Then, pipette slowly to resuspend the cells, avoiding top froth as much as possible. Place one-milliliter aliquots of the resuspended cells into 1.5-milliliter microtubes and keep them on ice or a pre-chilled cold block at four degree Celsius.
Next, sonicate each tube in a sonicator with a setup of 20%amplitude for three cycles and spin down the lysate at 12, 000 G for 10 minutes at four degree Celsius. After collecting the supernatant with a pipette, transfer to a 50-milliliter tube. Incubate the cell lysate at 37 degrees Celsius at 200 RPM agitation for 80 minutes.
After spinning down the lysate and collecting the supernatant in pre-chilled 1.5-milliliter microtubes, aliquot 30 microliters in pre-chilled PCR 8-strip tubes, while keeping all tubes on ice. Flash freeze the lysate aliquots on dry ice and store at minus 80 degrees Celsius. Prepare a master mix according to the buffer preparation tab as described in the manuscript.
For a reaction volume of 10.5 microliters, add 1.05-microliter of different Mg-glutamate stock concentrations and 9.45 microliters of the master mix and mix gently. Pipette 10 microliters of the above reactions into a 384-well square bottom microplate and cover it using an adhesive plate seal. Measure gene expression as fluorescence output by recording fluorescence data with a plate reader at regular intervals for eight hours of incubation at 30 degrees Celsius with continuous orbital shaking at 307 cycles per minute.
After identifying the Mg-glutamate concentration that results in the highest fluorescence value at the end point, proceed to K-glutamate calibration. Run the experiment and identify the highest fluorescence value from among the K-glutamate concentrations tested, thus identifying the optimal buffer composition for Mg-glutamate and K-glutamate for this lysate. Once the Mg-glutamate and K-glutamate values are established, prepare stock tubes of the optimized buffer composition according to the batch volume obtained.
After calculating the number of buffer tubes needed, aliquot 38 microliters per tube and store at minus 80 degrees Celsius. First, label a 1.5-milliliter microtube for the optimal master mix preparation. Add the correct volumes of the buffer and lysate and mix them gently.
Then, pipette the DNA samples first, followed by nuclease-free water, and finally, the optimal master mix. Mix the cell-free reaction gently with a pipette just before adding to the plate reader and avoid any bubbles. After setting up the reactions in the plate reader, kinetic runs recorded fluorescence data at regular intervals for eight hours of incubation at 30 degrees Celsius with continuous orbital shaking at 307 cycles per minute.
After calibration of the lysate, the Mg-glutamate optimal concentration was similar at eight millimolar across extracts for both linear and plasmid DNA. However, the optimal K-glutamate concentration for plasmid DNA is 140 millimolar, whereas the optimal K-glutamate concentration for linear DNA is 20 millimolar. After calibration steps, levels of GFP expressions were compared between linear and plasmid DNA in the wild-type, delta recB, and delta recBCD extracts.
The GFP expression from linear DNA reached 102%and 138%of the expression from plasmid DNA in extracts from delta recB and delta recBCD strains, respectively. The most important things in the protocol are sonication lysis of the cell and the buffer calibration steps separately for linear and plasmid DNA, particularly when using the native parameters. This technique would help speed up the testing of biological designs in cell-free systems, for example, the screening of biosensors, enzymes, and pathways, or prototyping of synthetic genetic circuits.
