Our method allows for the rapid characterization of genetic parts using cell-free expression systems and PCR instead of cloning. The main advantage of this technique is what typically takes days to weeks in cells can be done in hours to days using this method. Our protocol includes steps to make your own cell-free expression systems, which can be tricky in a number of places and may require adjustments depending on use case.
When first starting out, we recommend starting with commercial kits. Begin by preparing the PCR master mix according to the instructions in the manuscript and store it on ice. Aliquot 30 or 40 microliters of the master mix into the determined number of PCR tubes and add 10 microliters of each five micromolar variable primers to appropriately-labeled PCR tubes.
Place the PCR tubes into the thermocycler and run the PCR program as mentioned in the text manuscript. Then, hold the reactions at four degrees Celsius. If the original template is plasma DNA, add one microliter of DpnI restriction enzyme to digest the original template and incubate the reactions at 37 degrees Celsius for one hour.
Analyze five microliters of each PCR product by separating them using a 1%agarose gel at 180 volts for 20 minutes. Check for the correct band size, which will vary with the chosen core sequence and the length of the parts added. Purify the linear templates using a commercial PCR purification kit.
If multiple bands were present by gel electrophoresis, excise the bands of interest from the gel and purify the DNA using a commercial gel extraction kit, as per the manufacturer's instructions. Quantify each DNA template using a spectrophotometer, assessing its quality by checking that the 260 to 280 nanometer ratio is approximately 1.8. Thaw all the components on ice and prepare a master mix by mixing all the components thoroughly with a pipette, as mentioned in the manuscript.
Pay careful attention to avoid precipitation, especially for the amino acid mixture. Keep the master mix on ice. Chill a 384-well plate on ice and distribute the master mix in nine-microliter aliquots into each well.
Thaw the represser protein on ice and distribute it into an acoustic liquid handling source plate, ensuring that the appropriate amount of dead volume required for the type of source plate used is included. Distribute the represser protein in one-microliter volumes into the appropriate destination wells via the liquid handler. Include a serial dilution of the purified reporter or an appropriate chemical standard on the plate to compare results with other studies and other labs.
Choose a range of concentrations appropriate for the reporter used in the expected expression range of the experiments. Prewarm the plate reader to 30 degrees Celsius. If possible, on the instrument, set a one degree Celsius vertical temperature gradient to limit condensation on the seal.
Set the plate reader to read at settings appropriate for the reporter used in the core sequence, without shaking steps. Seal the 384-well plate with an impermeable plastic seal to prevent evaporation. Place the 384-well plate on the plate holder and begin reading.
15 linear templates, varying only in the distance of the T7 promoter relative to the tetO sequence, were prepared by PCR, amplifying the superfolder green fluorescent protein reporter, with primers designed to add each promoter variant. Analysis of data from a total of 540 reactions for the entire set of T7 tetO combinations showed that the T7 RNA polymerase downregulates T7-driven expression equally, up through 13 base pairs downstream from the start of the T7 transcript. More consistent dispensing across the series of eight destination wells using the acoustic liquid handler was observed when a five-microliter transfer was divided into separate one-microliter dispenses.
Our protocol can be used to rapidly screen genetic components, whether for screening of new biosensors or building of genetic parts'libraries.
