DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

The overall goal of the following experiment is to determine genomic binding sites of bacterial two component response regulators using an in vitro microarray based assay. This is achieved by cloning and purifying a tagged response regulator protein, activating it by in vitro phosphorylation and determining one gene target using electrophoretic mobility shift assays to use as a positive control for the downstream microarray steps. As a second step, the purified response regulator protein is mixed with she genomic DNA, which allows the protein bound DNA to be separated from input DNA using affinity purification.

Next, the protein bound DNA and input DNA are amplified QPCR verified for the enrichment of the positive control gene target in the protein bound fraction, fluorescently labeled, pooled and hybridized to a tiling microarray. In order to determine the genomic binding sites of the response regulator, the results show the most likely genes that are regulated by the response regulator under study and the results can be further validated using electrophoretic mobility shift assays. The main advantage of this technique over other methods like the in vivo chip chip, is that it can be used even if the activating signals and the conditions are not known for the two component systems.

This method is of great importance to the bacterial signaling field as a vast majority of response. Regulators have unknown gene targets and this method helps to unravel intricate regulatory networks. Demonstrating this procedure will be Amy Chen, a research assistant from my laboratory Prior to starting this procedure.

Purify an rr, specifically the DVU 3 0 2 3 protein expressed from a cloned DVU 3 0 2 3 gene mix. 0.5 picomoles of DVU 3 0 2 3 protein with 100 fent moles of a previously prepared by at inated DNA substrate in a buffered solution to a total volume of 20 microliters. After setting up a reaction without any protein as a control, incubate the reactions at room temperature on the bench for 20 minutes.

Following incubation, add five microliters of five x loading buffer to the binding reactions. Load 18 microliters of each reaction onto a previously prepared mini 6%poly acrylamide 0.5 XTBE gel and run at 100 volts for two hours after the gel has been run, cut a charged nylon membrane to the size of the gel and soak it in 0.5 XTBE for at least 10 minutes. Then remove the gel from the cassette.

Cut any ridges off the gel and sandwich the gel and membrane between two thick filter papers soaked in 0.5 XTBE. Place the gel and membrane inside a semi-dry blotting apparatus and run at 20 volts for 30 minutes. Following this, place the membrane inside a commercial UV light crosslinker instrument and set the time to three minutes after developing the blot using a chemiluminescent detection kit, image it using a computer hooked up to a CCD equipped camera.

Look for a shift in the DNA substrate mobility in the presence of the rr, which indicates that the RR binds the DNA being tested. Next, mix two to three micrograms of shared genomic DNA with 0.5 picomoles of DVU 3 0 2 3 in a buffered solution. After incubating the reaction at 25 degrees Celsius for 30 minutes, transfer 10 microliters of it to a 1.5 milliliter tube and label it as input DNA.

Add 30 microliters of nickel NTA aros resin to a 0.6 milliliter micro fuge tube. Centrifuge the sample at 100 GS for one minute to collect the resin at the bottom of the tube following centrifugation. Remove the supernatant at this point.

Add 100 microliters of wash buffer and flick the tube to mix the contents. After centrifugation, add 100 Gs for two minutes and removal of the supernatant. Add the remaining 90 microliters of the binding reaction to the wash to nickel NTA resin.

Then incubate the sample in a rotary shaker for 30 minutes. When finished, centrifuge the sample at 100 Gs for two minutes. After removing the supernatant, repeat the wash steps three more times.

Next at 35 microliters of elucian buffer to the resin and mix by vortexing. Incubate the sample on the bench at room temperature for five minutes. After centrifuging the sample, transfer the supernatant to a new 1.5 milliliter tube and label it as the protein bound DNA fraction.

Then add 35 microliters of the elucian buffer to the input DNA following purification of the input and protein bound DNA fractions. Add 10 microliters of each to separate PCR tubes. Then add one microliter of 10 x fragmentation buffer, two microliters of library preparation buffer and one microliter of library stabilization solution to each sample, mix the samples well by vortexing.

Eat the samples in a thermal cycler at 95 degrees Celsius for two minutes. When finished, chill the samples on ice. Next, add one microliter of library preparation enzyme to the samples and mix the contents by pipetting.

Incubate the samples in a thermal cycler. After incubation, add 47.5 microliters of water, 7.5 microliters of 10 x amplification, master mix, and five microliters of polymerase to each tube. Once the samples have been mixed, heat them in a thermal cycler.

Set up triplicate QPCR reactions for each DNA template by preparing a one x master mix for each previously designed primer set then aliquot 18 microliters of the master mix per well of a 96 Well PCR plate dilute the amplified and purified input and protein bound DNA samples with water to five nanograms per microliter and add two microliters of each sample to the wells. Following this. Seal the plate with an UltraClear QPCR sealing film.

Spin down the plate in a centrifuge at 200 GS for one minute. After placing the plate in a real-time QPCR machine cycle, using the following conditions to label input DNA with s si three and enriched DNA with sci five, mix one microgram of DNA with 40 microliters of S3 SCI-FI labeled nine mers, and adjust the volume to 80 microliters with water. Eat denature the samples at 98 degrees Celsius for 10 minutes in the dark in a thermal cycler.

When finished, quick chill the samples on ice for two minutes, add two microliters of three prime, five prime exo polymerase, five millimolar of deoxy nucleotide tric phosphates, and eight microliters of water to each reaction. After mixing, incubate the reactions at 37 degrees Celsius for two hours in the dark in a thermal cycler. At this point, add EDTA to 50 millimolar to stop the reactions and sodium chloride solution to 0.5 molar.

Then transfer to 1.5 milliliter tubes containing a 0.9 volume of isopropanol following incubation in the dark for 10 minutes. Centrifuge the samples at 12, 000 GS for 10 minutes. Pool together six micrograms each of the S3 and sci-fi labeled DNA in a 1.5 milliliter tube and vacuum dry in a centrifuge on low heat in the dark by covering the centrifuge lid if it is transparent.

Once the pellets are dry, we suspend them in five microliters of water. Then add 13 microliters of a previously prepared one x hybridization solution master mix to the samples after vortexing for 15 seconds, incubate the samples at 95 degrees Celsius in a dry bath for five minutes. Remove the samples from the dry bath and place them in a hybridization system at 42 degrees Celsius until ready for loading.

Next, place the previously prepared mixer slide assembly within the hybridization system. Load 16 microliters of sample into the fill port. Then seal the ports with an adhesive film and turn the mixing on in the system After hybridizing for 16 to 20 hours at 42 degrees Celsius.

Slide the mixer, slide into a disassembly tool and place it inside a dish containing warm buffer.One. Peel the mixer off while vigorously shaking the disassembly tool. Place the slide into a container with 50 milliliters of wash buffer one and shake vigorously for two minutes.

Then repeat the previous step with 50 milliliters of wash buffer two and 50 milliliters of wash buffer three RRR DVU 3 0 2 3 shifted. The upstream region of DVU 3 0 2 5 QPCR showed that the DVU 3 0 2 5 upstream region is enriched in the protein bound fraction relative to the input DNA, thus indicating that the binding conditions were appropriate for DVU 3 0 2 3 DVU 0 0 1 3 was a negative control. The top four peaks obtained after DAP chipp analysis were chosen as the most likely targets were DV 3 0 2 3.

The positive target DVU 3 0 2 5 was the first peak obtained with the highest score. Two gene targets are two other ly encoded lactate permease. The fourth gene target does not lie in an upstream region, but in the intergenic region between two convergently transcribed genes using the upstream regions of targets obtained by DAP chip meme was used to predict a binding site motif.

The motif was further validated by making substitutions in the conserved bases within the motif, which eliminated the binding shift. The validated motif was used to scan other sulfate reducing bacteria genomes that had DVU 3 0 2 3 orthologs loci were chosen as possible gene targets when the motif was located in upstream regions of open reading frames using motif sequences predicted for autologous rs. A consensus binding site motif was generated, which closely resembled the one for Divo Garris Hilton Burrow.

After watching this video, you should have a good understanding of how to use the Daptive method to determine gene targets for regulatory proteins. This method can be employed to analyze the binding size of any regulatory protein provided the protein can be purified and the genome sequence is known.