The overall goal of this procedure is to use yeast cells to find the biological processes targeted by a chemical of interest. This is accomplished by first determining the dose response profile of a chemical for yeast. The second step is to grow a complete collection of yeast gene knockout strains on a sub lethal dose of chemical and on a control plate.
Next yeast mutants that are hypersensitive to the chemical are identified. This list is bioinformatically analyzed to reveal all genes needed to tolerate the chemical. The final step is to perform a biological replicate of the experiment in low throughput.
This validates any chemical gene interactions found. Ultimately, this high throughput chemical genetic screening method is used to show which gene products and biological pathways are used to tolerate the chemical of interest. The main advantage of this technique over existing methods such as competitive growth of barcoded mutants in liquid culture is that no molecular biology techniques are required.
This method can help answer key questions in the field of chemical biology, such as how a novel small molecule can inhibit the growth of eukaryotic cells. To begin, prepare an overnight yeast culture by streaking out BY 4 7 4 1 cells on A-Y-E-P-D plate and incubate for 48 hours at 30 degrees Celsius or until visible colonies form. When the colonies are ready.
Prepare overnight liquid cultures by picking a single colony and inoculating five milliliters of YEPD liquid media in a sterile vessel. Then incubate overnight at 30 degrees Celsius with continuous rotation or shaking. Next, prepare solid agar media containing various doses of the chemical to be tested.
To do this. First, prepare several stock solutions of the chemical of interest at 100 times the final concentration in an appropriate solvent. Then for each concentration to be tested aliquot three milliliters of molten YEPD agar into several sterile culture tubes.
Base the tubes containing the agar into a 55 degrees Celsius water bath to prevent solidification of the agar. Now, for each concentration of chemical to be tested at 13, white release of the 100 x compound to an aliquot of molten agar tex, the tube for two to three seconds to mix and then pipette one milliliter from each tube into duplicate wells in a 12 world plate, allow the plate to set overnight at room temperature. The following day, inoculate 10 milliliters of YEPD liquid media with two microliters of the saturated overnight yeast culture.
Then pipette 25 microliters of this culture onto the chemical containing agar in the wells of the 12 whale plate and spread evenly using sterile glass beads or a sterile rod. Allow the plate to dry under a flame and then incubate the plate at 30 degrees Celsius for 48 hours. After the incubation time has elapsed, evaluate the total number and size of colonies in the wells.
Identify a sub lethal concentration of compound that does not inhibit growth by greater than 10 to 15%to use in the screen. Maintain the deletion mutant rate at a density of 384 colonies per plate by replicating on 16 YEPD agar plates, containing 200 microliters per milliliter of G four 18. Using a microbial arraying robotic system or manual pinning tools every three months.
Store the plates at four degrees Celsius until required. Then prepare 250 milliliters of YEPD agar media with the same concentration of G four 18 or five agar plates, and leave them to cool overnight at room temperature on a flat even surface. Remove the plates containing the deletion mutant collection from storage at four degrees Celsius and allow the plates to come to room temperature.
Use a delicate task wipe to remove any condensation that has formed on the lid of each plate to prevent water droplets being deposited on the array and cross contamination of the array mutants. Have the microbial array pinning robot condense the deletion mutant array from a density of 384 colonies per plate on 16 Petri dishes to 1, 536 colonies per plate on four Petri dishes. After incubating the array at 30 degrees Celsius overnight, examine the array to ensure uniform transfer of colonies from the source plates.
There will be several blank positions incorporated into the array, which serve as a guide that the deletion mutant array has been condensed correctly. These will be the source plates used to replicate onto media containing your compound of interest. A single source plate can be used for multiple pinnings.
Also, many robots have offset features which will allow, which will ensure a similar number of yeasts transferred each time. To set up the replica plate deletion mutant array repair, 700 milliliters of control vehicle only and experimental YEPD agar containing the chemical of interest. This is enough for performing the assay in triplicate.
Pour the plates using the control and experimental agar. Adding 50 milliliters to each plate. Allow the plates to cool at room temperature overnight on a flat, even surface, using the robot to replic a plate.
Inoculate the double mutant array onto three sets of plates containing chemicals at the experimentally determined concentration, as well as resets of plates containing the vehicle control. Incubate the plates on the bench top at room temperature for a total of 24 to 48 hours image. The plates are 24 and 48 hours of incubation by removing the lid and placing face down on a flatbed scanner.
Capture images at a resolution of at least 300 DPI. Alternatively, use a digital camera to capture images. If doing so, place plates face up on a dark background and remove the lids to image.
Then perform quantification and comparison of array colony sizes using one of several open source programs, including ball SGA tools and screen mill. At this stage, several yeast deletion mutants will score as hypersensitive to the chemical of interest streak out the desired hypersensitive mutants from the yeast deletion collection onto YEPD. Ager containing 200 micrograms per microliter of G four 18 incubate at 30 degrees Celsius until colonies form.
After verifying the genotype of the strain by diagnostic PCR and Agel electrophoresis inoculate five milliliters of the YPD liquid with each strain and incubate overnight at 30 degrees Celsius with rotation or shaking. Measure the OD 600 of each culture and dilute each strain to an OD 600 of one in sterile distilled water. These are now the normalized yeast cultures.
Next, dispense 100 microliters of normalized wild type by 4 7 4 1 yeast culture to well A one of a 96 well plate dispense 100 microliters of up to seven additional strains to wells, B one to H one. Next, dispense 90 microliters of sterile water to wells, A two to H two through a six to H six. Finally prepare a series of one to 10 dilution of normalized G sculptures.
Start by serially pipetting 10 microliters of column one to column two with a multi-channel pipette and continue across the 96 well plate until six dilution are made. Then use the multi-channel pipette to two to five microliters of the diluted yeast cultures in a grid on YEP D's, solid media containing chemical and vehicle control. Incubate the plates at the appropriate temperature for 24 to 48 hours following the incubation.
Inspect the plates and compare the sensitivity of select mutants to chemical relative to the BY 47 41 control image plates at 24 and 48 hours. As before the following images show results from the procedure to determine the subin inhibitory concentration of chemical. The first image shows growth of a saturated BY 4 7 4 1 culture diluted one to 5, 000 and plated on increasing concentrations of five fluoro uracil.
The 10 micromolar concentration is selected as a sub-in inhibitory dose. This image shows the growth curve of BY 4 7 41 cells in liquid media containing five fluro uracil. Approximately four times 10 to the four cells were deposited in triplicate and increasing concentrations of five fluoro uracil and optical densities were measured every 10 minutes for 22 hours.
Again, it can be seen that the 10 micromolar dose is the subin inhibitory dose. These next images show the results from a chemical genetics green of five fluoro uracil. Here, a S sachar visier deletion mutant array replicated on YPD agar containing 10 micromolar five Fluor uracil on the right and DMSO control on the left is shown.
This graph shows the results of the chemical genetics green, the relative growth of mutants on 10 micromolar. Five Fluor uracil compared to DMSO control are ordered by a ray position here. Relative growth of mutants on 10 micromolar, five flora uracil compared to DMSO control are ordered by colony size ratio.
A ratio threshold of less than 0.8 is indicated on both graphs. This final image shows results of three validation assays. Serial dilutions of several isolated mutant strains were grown on DMSO control on the left, 10 micromolar, five fluoro UIL in the center and 0.015%methyl methane sulfonate on the right.
While attempting this procedure, it is important to ensure you're working with a reliable copy of the deletion mutant array as well to validate any results using an independent methodology such as yeast spotting assays or yeast tetrad dissection. This technique provides an efficient means for scientists in the field of small molecule research to explore a compound's mode of action using systems level approaches in budding yeast.
