Paarung-basierte Überexpression Bibliothek Screening in der Hefe

This method can help answer key questions in the neurodegenerative disease field, such as what genetic factors and pathways regulate the toxicity of disease-associated proteins. The main advantage of this technique is that the highly efficient process of yeast mating is used to screen yeast models against a large collection of library genes. Though this method can provide insight into the toxicity of neurodegenerative disease proteins, it can also be applied to the study of other cellular processes in yeast, such as tolerance of other stress conditions.

Begin this procedure by aliquoting five microliters per well of plasmid DNA from an arrayed plasmid library to round bottom 96-well plates. Keep the plates at four degrees Celsius. Next, inoculate 150 milliliters of yeast peptone dextrose or YPD medium in a 500 milliliter flask with a colony of the haploid yeast strain W303 Alpha.

Incubate the culture at 30 degrees Celsius with shaking at 200 rpm overnight. On the following morning, after measuring the OD600 of the overnight culture, dilute the culture to an OD600 of 0.1 in two liters of YPD. Incubate at 30 degrees Celsius with shaking at 200 rpm for about five hours, until the culture reaches an OD600 of 0.4 to 0.6.

To harvest the yeast culture, fill eight sterile 250 milliliter centrifuge bottles and centrifuge at 3000 xG for 10 minutes at room temperature. Pour off the supernatant without disrupting the pellet. Wash the yeast as described in the text protocol, and consolidate the cells into two centrifuge tubes.

Resuspend each cell pellet in 25 milliliters of a 0.1 molar lithium acetate solution. Combine the resuspended cells, add five milliliters of salmon sperm DNA, and transfer them to a 150 milliliter flask. Incubate at 30 degrees Celsius with shaking at 225 rpm for 30 minutes.

After 30 minutes, pour the cell mixture into a sterile disposable reagent reservoir. Using a multichannel pipette, transfer 35 microliters of the cell mixture to each well of the round bottom 96-well plates, containing the library plasmid DNA prepared earlier. Vortex the 96-well plates at 1000 rpm for one minute.

Incubate the plates at 30 degrees Celsius without shaking for 30 minutes. Do not stack the plates, so the heat can transfer more efficiently. Remove the 96-well plates from the 30 degree Celsius incubator and mix them at 1000 rpm for 30 seconds.

Add 125 microliters of transformation buffer to each well, and then vortex the plates at 1000 rpm for one minute. Incubate the plates at 30 degrees Celsius for 30 minutes. Then heat shock the yeast by placing the plates in a 42 degree Celsius incubator for 15 minutes.

Centrifuge the plates at 3000 xG for five minutes. Remove the transformation buffer from the wells by inverting the plates over a waste bin and forcefully dumping the buffer from the plates. Quickly blot the inverted plates on a clean paper towel to remove any liquid on top of the plates.

Rinse the cells by adding to each well, 200 microliters of minimal dropout medium corresponding to the selectable marker on the library plasmid. Synthetic ura minus medium is used here. Centrifuge the plates at 3000 xG for five minutes.

Remove the supernatant over a waste bin as demonstrated earlier. Add 160 microliters of minimal ura minus medium, containing 2%glucose to each well and vortex the plates at 1000 rpm for one minute. Incubate the plates without shaking at 30 degrees Celsius for 48 hours.

After 48 hours, a pellet of transformed yeast should be visible at the bottom of each well. Using a liquid dispenser, add 100 microliters of ura minus medium containing glucose into each well of a new set of 96-well plates. Vortex the plates containing the transformed yeast at 1000 rpm for 30 seconds.

Using a sterile plastic 96-pin replicator, place the pins into the wells containing the transformed yeast and then inoculate them into the corresponding wells of the new plates filled with media. Incubate the new plates at 30 degrees Celsius for 24 hours. After 24 hours, a pellet of yeast should be visible at the bottom of each well that contains successfully transformed yeast.

To save these yeast strains as glycerol stocks, add 50 microliters of 50%glycerol to each well, vortex the plates at 1000 rpm for 30 seconds, seal the plates with aluminum foil, and freeze at minus 80 degrees Celsius. To revive the library strains from the glycerol stock, take the 96-well plates out of the minus 80 degrees Celsius freezer, remove the aluminum foil, and let the yeast thaw at room temperature for approximately 30 minutes. Once the yeast has thawed, use a sterile plastic 96 pin replicator to inoculate the glycerol stocks to 160 microliters of fresh ura minus media containing 2%glucose in 96-well plates and incubate at 30 degrees Celsius for 24 hours.

Immediately after the library strains are used, seal the plates, and return them to the minus 80 degrees Celsius freezer. On the same day that the library strains are thawed, inoculate the query yeast strain in 50 milliliters of YPD and grow at 30 degrees Celsius with shaking at 250 rpm overnight. On the following morning, pour the query yeast strain into a sterile disposable reagent reservoir and aliquot 160 microliters of the query strain to each well of a 96-well plate.

Using the liquid dispenser, dispense 160 microliters per well of YPD media into 96-well plates. Briefly vortex the 96-well plates containing the query strain and the library strain plates and then use a sterile 96-pin replicator to transfer the library strains to the YPD plates that have been inoculated with the query strain. Incubate the YPD plates at 30 degrees Celsius for 24 hours.

After 24 hours a pellet of yeast will be visible at the bottom of each well. Fill 96-well plates with minimal droupout medium containing 2%Raffinose corresponding to the selectable markers on the plasmid in the query strain, as well as on the library plasmid. Use a sterile 96-pin replicator to transfer yeast from the YPD mating cultures to the selective medium.

Incubate the 96-well plates at 30 degrees Celsius for 48 hours. Only yeast cells that have mated and formed diploid cells and therefore contain both the query plasmid and the library plasmid will be able to grow in this medium. After 48 hours of growth in the Raffinose containing selective medium, observe that a pellet is visible at the bottom of the wells that contain diploid cells.

Vortex the 96-well plates at 1000 rpm for one minute. Use a robotic spotting machine to spot the yeast on agar plates in quadruplicate to ura-his-droupout plates containing 2%galactose and 2%agar, and to ura-his-droupout plates containing 2%glucose and 2%agar. After spotting, let the agar plates dry and then place them upside down in a 30 degrees Celsius incubator for four days.

Every 24 hours, photograph the agar plates to record the growth of the yeast. Toxicity of the ALS associated protein FUS was first examined in diploid yeast. As indicated by the agar plate containing galactose, which induces FUS, FUS toxicity is consistent in both diplod and haploid yeast.

Five genes previously identified as suppressors of FUS toxicity from a transformation based method was tested by the mating method. Lane one is a control yeast strain transformed with two empty vectors. Lane two is the diploid FUS yeast strain with an empty vector where the expression of FUS is very toxic.

Lanes three to seven show diploid yeast expressing FUS as well as a suppression gene. All five genes rescue the toxicity of FUS in diploid yeast, indicating that the mating method was effective. This mating based method was then applied to an over-expression library screening of 940 genes.

And a photo of one representative plate is shown. As indicated on the galactose plate, where FUS and library genes are expressed, FUS was toxic to the diplod yeast cell. The library gene, indicated by the green square, rescued FUS toxicity.

While the library gene, indicated by the red square, enhanced toxicity. Once mastered, this technique can be done in 120 hours, if it is performed properly. While attempting this procedure, it is important to remember to check beforehand that the phenotype used for screening is not haploid mating type dependent.

After watching this video, you should have a good understanding of how to identify genetic factors upon overexpression that rescue the toxicity of disease associated proteins in yeast. After its development, this technique can pave the way for researchers in the field of neurodegenerative disease, to explore mechanisms of cytotoxicity of disease associated proteins in the yeast model. Following this procedure, other methods such as the beta galactosidase assay can be performed in order to answer additional questions, like whether the rescue is specific.