The overall goal of this experiment is to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomic study in a regular laboratory setting. This method can help answer key questions in functional genomics field, such as planned genomics. The main advantage of this technique is that it detects protein DNA interactions involving more than one protein or transcription factor.
Begin this procedure on the afternoon of what will be designated as day one. Start a 5 milliliter culture in synthetic defined or SD medium without uracil from a freshly streaked Petri dish. Incubate overnight in a 30 degree Celsius shaker.
On the following afternoon, inoculate 10 milliliters of YPDA medium with 500 microliters of the overnight culture and incubate overnight in a 30 degree Celsius shaker. Early in the morning of the following day, inoculate 100 milliliters of YPDA medium with 2 milliliters of the overnight culture. Grow this fresh culture at 30 degrees Celsius until the optical density at 600 nanometers, reaches 0.4 to 0.8.
Centrifuge at 1000 times G and 21 degrees Celsius for 5 minutes. Discard the supernatant. Add 10 milliliters of sterile water and reconstitute the pellet by vortexing.
Centrifuge at 1000 times G and 21 degrees Celsius for five minutes. Discard the supernatant, add 2 milliliters of TE lithium acetate, and reconstitute the pellet by vortexing. Centrifuge at 1000 times G and 21 degrees Celsius for five minutes.
Discard the supernatant. Add 4 milliliters of TE lithium acetate plus 400 microliters of salmon sperm DNA and resuspend the pellet by pipetting up and down. The cells are now ready for transformation as demonstrated in the next segment.
Co-transformation of the cells is performed in a 96 well U-bottom mixing plate. First, distribute 20 microliters of competent cells per well in the 96 well plate. Then, add to the wells, 150 nanograms each of the two plasmids and the normalization control.
This schematic shows the general plate set up for the co-transformation of the yeast cells with protein of interest. The plate set up can be modified according to the need of the experiment and number of DNA regions or fragments tested. Add 100 microliters of TE lithium acetate polyethylene glycol per well and mix three times by pipetting.
Cover the plate with a breathable seal and incubate at 30 degrees Celsius for 20 to 30 minutes. Heat shock at 42 degrees Celsius for precisely 20 minutes. After the heat shock, centrifuge at 1000 times G and 21 degrees Celsius for 5 minutes.
Use a multichannel pipette to remove the supernatant. Add 110 microliters of TE and mix. Centrifuge again for 5 minutes.
Remove 100 microliters of the supernatant from each well. Immerse a puncher in 100%ethanol, flame it, wait for one minute for the puncher pins to cool, and then use the sterile puncher to resuspend the pellet. Transfer cells onto SD tryptophan and lucine drop out auger plates.
Incubate the plates at 30 degrees Celsius for two days. On the afternoon of day five, retrieve the auger plates from the incubator. Fill each well of the sterile 96 well u-bottom mixing plate with 50 microliters of TE.Prewet a sterile puncher in TE, punch the colonies from the SD tryptophan and lucine drop out plate, and resuspend them in the TE filled plate.
Transfer the colonies onto new SD tryptophan and lucine drop out auger plates, for optimal surface coverage. Incubate the plates at 30 degrees Celsius for 2 days. On the afternoon of day 7, retrieve the auger plates from the incubator.
Fill each well of a sterile 96 well u-bottom mixing plate with 50 microliters of SD tryptophan and lucine drop out medium. Fill each well of a sterile 96 deep well block with 180 microliters of SD tryptophan and lucine drop out medium. Sterilize the puncher, prewet the puncher and medium, and transfer colonies from the plate to the wells that each contain 50 microliters of medium.
Transfer 20 microliters of yeast to the 180 microliters of SD tryptophan and lucine drop out medium. Seal the block with a breathable seal, and incubate at 30 degrees Celsius with shaking for 36 hours. On the morning of day nine, transfer 100 microliters of culture to 500 microliters of YPDA medium in a sterilized 96 deep well block.
Cover with a breathable seal and incubate at 30 degrees Celsius with agitation at 200 rpm for three to five hours. After three to five hours, resuspend the culture and transfer 125 microliters from each well to a spectrophotometer plate. Measure the optical density at 600 nanometers to ensure it is between 0.3 and 0.6.
Centrifuge the remaining cells in the deep well block at 3000 times G and 21 degrees Celsius for 10 minutes. Remove the supernatant by inverting. Add 200 microliters of Z buffer to each well and vortex.
Centrifuge at 3000 G and 21 degrees Celsius for five minutes. Remove the supernatant by inverting. Add 21 microliters of Z buffer to each well and vortex.
Cover the plate with sealing foil that is resistant to freeze thaw cycles. In a fume hood, perform 4 cycles of freeze/thaw using liquid nitrogen and a 42 degree Celsius water bath. Add 200 microliters of freshly prepared Z buffer beta mercaptoethanol ONPG solution to each well.
Incubate at 30 degrees Celsius for 17 to 24 hours or until color develops. On the morning of day 10, check that the color has developed. Add 110 microliters of one molar sodium carbonate to each well to stop the reaction.
Record the time and vortex the plate. Centrifuge at 3000 times G and 21 degrees Celsius for ten minutes. Use a multichannel pipette to transfer 125 microliters of supernatant from each well to a spectrophotometer plate.
Measure the optical density at 420 nanometers. Calculate beta galactosidase activity in a spread sheet as described in the text protocol. In this study, the promoter region of 2600 base pairs upstream from the start of the transcription start site was segmented into six regions or fragments.
This photo is an example of a well-grown and positive plate after day five of the protocol. In this representative result, DNA fragments one and four show positive interaction with the protein A and B complex. Upon the measurement of beta golactisidase activity, fragment one shows four fold induction of the reporter gene activity.
It is important to remember that this procedure is recommended for obtaining information on DNA regions only after the confirmation of the proposed protein protein interaction and is not designed to provide information of target sites. Following this procedures, other methods, such as chip assay and Msar can be preformed in order to answer additional questions. For example, identifying the target sites in vivo.
After watching this video, you should have a good understanding of how to test and validate the role of multimeric protein complexes binding to a common DNA target.
