This protocol is significant because it details how to quickly generate large amounts of DNA template for use in cell-free reactions from a small gene fragment received from a synthesis facility. Rolling circle amplification can generate large amounts of DNA faster than traditional cloning. So it supports higher throughput design build test learn cycles from libraries of synthetic DNA.
These techniques can have large inherent variability for new users due to the many steps and small volumes required. Careful attention should be given to the technique, and practice makes perfect. To begin, add all the components from a PCR kit in a single PCR tube.
Then add one microliter of the resuspended gene fragment. Gently homogenize the mixture by vortexing on a medium setting for five to 10 seconds. After programming the thermocycler according to the kit manufacturer's protocol, run the PCR.
Upon completion of the PCR, use a PCR cleanup kit to purify the DNA from the reaction mix. In a 1.5 milliliter tube, add the DNA binding buffer and the PCR sample at a five to one ratio. Transfer this mixture to the spin column and centrifuge at 16, 000 times G for one minute.
Discard the flow through. Add 200 microliters of the DNA wash buffer to the column and incubate it at room temperature for one minute. Centrifuge the column again and discard the flow-through.
Wash the column once more with the DNA wash buffer, as demonstrated. After discarding the flow-through from the second wash, remove any remaining buffer by centrifuging the column for an additional one to two minutes. To elute the DNA, transfer the column into a new 1.5-milliliter tube.
Then add 46 microliters of double-distilled water into the column. After a one-minute incubation, collect the DNA into the tube by centrifugation. And quantify the purified DNA using a spectrophotometer.
To digest and circularize the DNA, combine five microliters of the necessary reaction buffer, 20 units of the HindIII restriction enzyme, and 45 microliters of the purified DNA in a PCR tube. Using a pipette, gently homogenize this mixture. Then incubate it in a thermocycler for 15 minutes at 37 degrees Celsius.
After the incubation is complete, heat inactivate the HindIII enzyme by incubating for 20 minutes at 80 degrees Celsius. Then add five microliters of T4 Ligase buffer and 800 units of T4 Ligase to the newly digested DNA. After gently mixing with a pipette, incubate the mixture for one hour at 25 degrees Celsius to perform the circularization reaction.
Once the reaction is complete, purify the DNA using a PCR cleanup kit as demonstrated previously. Then quantify the DNA. To perform the rolling circle amplification, combine all reaction components and one microliter of the purified circularized expression template in a single tube.
Homogenize the mixture with a pipette and aliquot 10 microliters of the mixture into four separate tubes. Incubate the tubes at 30 degrees Celsius for four to 18 hours, then heat inactivate the enzyme by incubating at 65 degrees Celsius for 10 minutes. After heat inactivation, encourage condensation at the bottom of the tube by incubating at 12 degrees Celsius for five minutes.
Next, dilute the resulting solution by adding 15 microliters of double-distilled water to each tube. Combine the contents of each tube before purifying and quantifying the DNA as demonstrated previously. To perform the cell-free reaction, add the various required components into a tube and dilute to the final desired volume with double-distilled water.
Mix the solution thoroughly by pipetting half the solution up and down 10 to 20 times. Transfer the reaction mixture in 15-microliter aliquots to the desired wells in the microtiter plate. Then seal the plate with a colorless sealing film to maintain humidity and prevent evaporation.
Place the sealed plate in the plate reader and allow the reaction to proceed to completion. If expressing the subtilisin BPN gene using the cell-free system, aliquot 94 microliters of double-distilled water and one microliter of 10 micromolar PNA in a flat-bottom, colorless 96 well plate. Then add five microliters of the completed cell-free reaction and read the plate using a plate reader set to measure absorbance at 410 nanometers every 20 seconds for 10 minutes while maintaining a temperature of 25 degrees Celsius.
Expression of the Superfolder GFP in the BL21 DE3 star extract using only 3 microliters of unpurified RCA DNA in a 15-microliter reaction is comparable to that of the PJL1 plasmid. Doubling and tripling the amounts of template appears to offer no obvious benefit, suggesting already saturated levels of the template at 3 microliters per reaction. Conversely, there appears to be a benefit to increasing the amount of RCA template when using the shuffle strain cell extract.
Proteins requiring lower temperatures or longer folding times affect the time required to complete the entire workflow. For example, assaying subtilisin after four hours of expression leads to a failed result, as four hours is not enough for subtilisin maturation. However, allowing the reaction to continue to 16 hours leads to detectable levels of subtilisin.
All components needs to be well-mixed for this protocol to work at maximum efficiency. Following these methods, a large library of protein candidates can be generated synthetically and then expressed at sufficient yield for characterization. This method paved the way for our group to develop N C two sensors that can work in cell extracts, allowing us to quickly characterize the prototype protein.
This will enable us to more rapidly and intelligently iterate on protein design of new bio catalysts and therapies.
