Our method is one of the first to be able to dissect the genetic bases of trait differences on a genome-wide scale between species instead of within species. The main advantages of this technique are that it is high-throughput, high resolution, and widely applicable. To begin this procedure, retrieve the JR507 freezer stock strain from storage at minus 80 degrees Celsius, and streak it out to single colonies on a YPD agar plate.
Incubate at 26 degrees Celsius for two days or until colonies appear. Then inoculate 100 milliliters of liquid YPD in a 250 milliliter glass flask with a single colony of JR507, and incubate at 28 degrees Celsius while shaking at 200 RPM for 24 hours or until stationary phase is reached. The next day, measure the od600 of the overnight culture.
Back dilute some of the overnight culture with fresh liquid YPD into a new one liter flask to create a new culture with an od600 of zero point two and a volume of 500 milliliters. Repeat this process three more times to create a total of four cultures with an od600 of zero point two, using the same overnight culture for all of the new cultures. Incubate all of the cultures at 28 degrees Celsius for six hours while shaking at 200 RPM.
After this, combine two of the 500 milliliter cultures to create a one liter culture. Combine the remaining two 500 milliliter cultures to create a second one liter culture. First, split each of the one liter cultures into 20 50 milliliter aliquots each in a plastic conical tube for a total of 40 tubes.
Set aside 20 tubes. Centrifuge the remaining 20 tubes at 1000 times G for three minutes to pellet the yeast cells. Discard the supernatant, and resuspend each pellet with 25 milliliters of sterile water by vortexing.
Centrifuge at 1000 times G for three minutes. Discard the supernatant, and resuspend each pellet with five milliliters of 1X Tris-EDTA zero point one Molar lithium acetate buffer by vortexing. Centrifuge at 1000 times G for three minutes.
Discard the supernatant, and resuspend each pellet again with five milliliters 1X Tris-EDTA zero point one Molar lithium acetate buffer by vortexing. Centrifuge at 1000 times G for three minutes. While the cells are centrifuging, prepare at least 120 milliliters of a solution containing polyethylene glycol, lithium acetate, and Tris-EDTA buffer.
Store this solution on ice. To prepare the plasma DNA for transformation, first boil four milliliters of salmon sperm DNA at 100 degrees Celsius for five minutes. After this, immediately cool it on ice for five minutes.
Mix 20 milliliters of pJR487 at a concentration of 538 nanograms per milliliter with four milliliters of the cold salmon sperm DNA. Keep this mixture on ice until ready to use. Next, add 600 microliters of the plasma and sperm DNA mixture on top of each cell pellet.
Add three milliliters of the PEG lithium acetate TE mixture to each pellet. We suspend by pipetting up and down and then vortexing. Incubate the tubes at room temperature for 10 minutes.
Then heat shock each tube in a water bath at 39 degrees Celsius for 26 minutes, making sure to invert each tube every few minutes. Centrifuge the tubes at 1000 times G for three minutes. Discard the supernatant, and resuspend each pellet in 10 milliliters of YPD by vortexing.
After this, combine all 20 tubes into a new glass flask. Add 433 point four milliliters of liquid YPD into a new one liter glass flask. Then transfer 66 point six milliliters of cells into the flask.
Repeat this transferring process two more times to use the entire volume of transformed cells. Then measure the od600 of each new 500 milliliter culture. Incubate all three cultures at 28 degrees Celsius for two hours while shaking at 200 RPM.
After this add zero point five milliliters of 300 milligrams per microliter G418 to each of the flasks to a final concentration of 300 micrograms per milliliter. Return the flasks to the shaking incubator at 28 degrees Celsius and 200 RPM. Repeat this entire process with the remaining 20 conical tubes.
At this point, there should be six one liter flasks, each containing 500 milliliters of cells with G418 added. Incubate all six flasks at 28 degrees Celsius with shaking at 200 RPM for approximately two days or until an od600 of approximately two point three is reached in each flask. Then, combine all six flasks together to create a single culture.
Use this combined culture to inoculate two new one liter flasks with 500 milliliters of YPD and G418 to an od600 of zero point two. Incubate these new cultures overnight at 28 degrees Celsius with shaking at 200 RPM until it reaches an od600 of approximately two point two. After this, combine both cultures into a single culture, and measure the od600 of the combined culture.
Centrifuge 25 milliliters of this culture at 1000 times G for three minutes. Calculate the number of total od600 units of cells that are in the 25 milliliters as outlined in the text protocol. Discard the supernatant, and resuspend the cells in enough water to create a cell suspension with an od600 of one point eight five per milliliter.
Using glass beads, plate one milliliter of resuspended cells onto each of 12 large, square complete synthetic agar plates with 5-FOA. Incubate each plate at 28 degrees Celsius for one to two days, or until a lawn forms on the plate. Next, use small sterile squeegees to scrap the cells off of half of the plate, and into a tube, containing 35 milliliters of sterile water.
Repeat this process for the remaining six plates, resulting in two tubes of cells in water. Combine the cell suspensions into a single flask, and measure the od600 of the suspension, using water as a blank. Use water to adjust the cell concentration until the od600 reaches 44 point four per milliliter.
Next, prepare freezer stocks of cells and store them at minus 80 degrees Celsius as outlined in the text protocol. In this study, S.Cerevisiae and S.Paradoxus are mated to form a sterile hybrid, which is subjected to transpose on mutagenesis. Each mutagenized clone is a hemizygote, a diploid hybrid in which one allele of one gene is disrupted.
The hemizygotes are competed at high temperature, and the DNA is isolated from each culture. The DNA is shirred and ligated to adapters, and the junction between the transposon and the genome is amplified with a transposon-specific primer and an adapter-specific primer. Counting up bulk sequencing read counts from the amplicon, report the fitness of a clone in the population.
With the results in hand from this sequencing, the hemizygote abundances for a given gene, can be compared at the two temperatures between two classes of hemizygotes. Clones where only the S.Cerevisiae allele was wild type and functional, and clones relying only on the S.Paradoxus allele. In this representative implementation, strong signals are detected at eight housekeeping genes.
In each case, transposon insertions in the S.Cerevisiae allele in the hybrid, compromised growth at high temperature. These low-si represented candidate determinants of the thermotolerance trait that distinguishes S.Cerevisiae from S.Paradoxus. Now that we have completed this experiment, we can dissect other traits that differ between these species, like cold or salt tolerance.
