Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

The overall goal of this procedure is to derive DNA strand displacement gates from bacterial plasmids. The main advantage of this technique is that it utilizes bacterial plasmid as a highly pure source to generate robust DNA gates. Demonstrating this procedure will be my colleagues Sam Dip and myself To begin mass produce and then isolate containing DNA gates by using DNA isolation kits as described in the accompanying text protocol and in accordance with the manufacturer's instructions following ellucian.

Measure the concentration of plasmid DNA using standard techniques. Then digest the DNA by adding four units of the restriction enzyme PV U2 HF for each milligram of the plasmid. Next, add two equivalent volumes of ice cold absolute ethanol to the sample.

Incubate the mixture at minus 80 degrees Celsius for at least one hour to precipitate the DNA. Then pellet the precipitated DNA by centrifuging the sample at 10, 000 to 14, 000 times G and zero degrees Celsius for 30 minutes. Remove the SNAT and add 1000 microliters of room temperature 95%ethanol to the sample.

Then invert the sample 10 to 15 times next, centrifuge the sample at 10, 000 to 14, 000 times G and four degrees Celsius for 10 minutes. When finished, remove the supernatant and air dry the DNA on a bench for 10 to 20 minutes. When removing the supernatant from the precipitated DNA, be careful not to disturb the pellet or the yield will be significantly decreased.

Once dry re suspend the DNA pellet in up to 200 microliters of nuclease free water and vortex to mix. Adding more than 200 microliters of water will generally make the sample to dilute for use. In kinetics experiments, measure the resuspended DNA using a spectrophotometer following the manufacturer's instructions.

Then add four units of the nicking enzyme M-B-B-S-R-D one per one microgram of plasmid, and add the corresponding enzyme buffer. Incubate the sample at 65 degrees Celsius for one hour to digest the joint gates. Next, digest the fork gates by adding eight units of the nicking enzyme NT BST mb one per one microgram of plasmid and the corresponding enzyme buffer.

Incubate the sample at 55 degrees Celsius for one hour. To prepare the fluorescent reporters first Resus, suspend and quantitate the samples as described in the accompanying text protocol. Then mix 10 microliters of the reporters'bottom strand with 13 microliters of the top quencher strand in tris acetate EDTA buffer with 12.5 millimolar of magnesium.

To ensure that all flora, four labeled strands are quenched, a 30%excess of the quencher labeled strand should be added. Next, anil the reporter C complex using a thermal cycler. Cool the sample from 95 degrees Celsius to 20 degrees Celsius at a rate of one degree Celsius per minute.

When finished, stored the sample at four degrees Celsius following calibration. Begin the fluorescence measurements by first setting the temperature controller to 25 degrees Celsius while the temperature stabilizes. Set up the proper parameters for kinetics measurements in the data acquisition software.

After opening up the software for the spectral fluorimeter, set the slip width to 2.73 nanometers for both the excitation and emission monochrome. Then set the integration time to 10 seconds for every 62nd time point and set the total measurement time to 24 hours. Lastly, set the excitation and emission wavelengths to match the flora forest used in the experiment.

Next, add 410.2 microliters of nuclease free water and 52.8 microliters of 10 extras acetate EDTA buffer containing 125 millimolar of magnesium to a synthetic quartz cell. Also add two microliters of 300 millimolar poly T strands, and then vortex the synthetic quartz cell for 10 to 15 seconds. Next at nine microliters of the reporter strands at 10 micromolar and six microliters of each auxiliary strand at 10 micromolar.

Then at 45 microliters of both the joint and fork gates at one micromolar and gently mix the solution by pipetting it up and down at least 20 times. Finally, at nine microliters of 10%sodium do ductal sulfate to achieve a final concentration of 0.15%SDS gently mix the reaction by pipetting up and down at least 20 times immediately place the synthetic quart cells into the chamber of the spectra fluter and start the kinetics measurement. After five minutes of measurement, add three microliters of the input strands.

Add 10 micromolar to the synthetic quart cell. While the data acquisition program is paused, gently mix the reaction by pipetting it up and down at least 20 times, and then close the light cover and continue to record the reaction kinetics until it reaches steady. State kinetics data for the plasma derived gates and the synthesized gates are shown here in the experiments.

The concentration of signal strand A is fixed while the amount of the catalytic signal B is varied. Signal C is used to read out the progress of the reaction without interrupting. The catalytic cycle turnover is defined as the amount of signal C produced for each catalyst B at a given time.

For an ideal catalytic system, this turnover number should linearly increase with time and be independent of the amount of catalyst as long as the substrate is not limiting. Here it is observed that the synthesized system deviates from the ideal linear increase of turnover much earlier than the plasma derived system does, indicating sequestration of the catalyst through an undesirable side reaction. The circuit leakage of both the plasma derived and the synthesized gates are compared here, and it is observed that the ratio of leak signal using plasma derived gates is about 8%less than that using synthesized gates after 10 hours of reaction.

After watching this video, you should have a good understanding of how to generate robust DNA gates from bacterial plasmid and test the gates using fluorescence kinetics measurements.