Name: Mating-based Overexpression Library Screening in Yeast (Video) | JoVE Journal
Uploaded: 2018-07-06T14:00:00.000+00:00
Duration: 11 min 39 s
Description: Learn a mating-based overexpression screening method in budding yeast using an arrayed plasmid library. This technique efficiently identifies genetic factors that regulate toxicity of neurodegenerative disease proteins.

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5 minutes

We are thankful for the thoughtful discussions with members of the Ju laboratory and Zhong laboratory, and the financial support from the Wright State University.

NOTE: The protocol described here is designed for screening library plasmids contained in ten 96-well plates but can be scaled up or down accordingly. The protocol needs to be repeated to complete the whole library screening. Usually, screening against 10 plates of library genes each time can be comfortably handled by 1 person.
1. Preparation for 96-well Yeast Transformation
NOTE: This step is done as previously described7,10.
<ol>
	<li>Aliquot 5 &#181;L (about 50&#8211;100 ng) of plasmid DNA from an arrayed plasmid library into each well of a round-bottom 96-well plate. 
	NOTE: An example of such a library would be a yeast gene overexpression library10.</li>
	<li>Inoculate 150 mL of yeast peptone dextrose (YPD) media in a 500 mL flask with a colony (2&#8211;3 mm diameter) of the haploid yeast strain W303&#945;. Incubate them overnight at 30 &#176;C with shaking (200 rpm).</li>
	<li>The following morning, measure the OD600 of the overnight culture and dilute the yeast culture to OD600 = 0.1 in (up to) 2 L of YPD. Incubate it at 30 &#176;C with shaking (200 rpm) for ~5 h until the culture reaches OD600 = 0.4&#8211;0.6.</li>
</ol>
2. Yeast Transformation
<ol>
	<li>Harvest the yeast culture by filling 8 sterile 250 mL centrifuge bottles and centrifuging them at room temperature at 3,000 x g for 10 min. Pour off the supernatant without disrupting the pellet. 
	NOTE: Carry out all centrifugation steps at room temperature.</li>
	<li>Wash the yeast with sterile-distilled H2O. For this, add 100 mL of sterile H2O to each centrifuge bottle and vortex it to resuspend the cell pellet. Combine the washed cells into 2 bottles and centrifuge them at 3,000 x g for 5 min. Pour off the supernatant.</li>
	<li>Wash the cells in each bottle in 100 mL of 0.1M LiOAc/1XTE (100 mM LiOAc; 10 mM Tris, pH 8.0; 1 mM EDTA) and centrifuge them at 3,000 x g for 5 min. Pour off the supernatant.</li>
	<li>While centrifuging, boil 5 mL of salmon sperm DNA (10 mg/mL) at 100 &#176;C using a block heater for 3 min and then cool it on ice.</li>
	<li>Resuspend the cell pellet in 25 mL of 0.1M LiOAc/1XTE in each bottle, combine the resuspended cells and transfer them to a 150 mL flask. Add 5 mL of the pre-cooled salmon sperm DNA and incubate it at 30 &#176;C with shaking (225 rpm) for 30 min.</li>
	<li>Pour the cell mixture into a sterile disposable reagent reservoir and, using a multichannel pipette, transfer 35 &#181;L of the cell mixture to each well of the round-bottom 96-well plate containing the library plasmid DNA. Vortex the 96-well plates using a plate vortexer for 1 min at 1,000 rpm. Incubate the plates for 30 min at 30 &#176;C without shaking. 
	NOTE: Do not stack the plates, so the heat can transfer more efficiently. We have found that vortexing the 96-well plates at 1,000 rpm does not cause liquid to spill out of the wells, but a safe vortex speed should be tested before performing the step.</li>
	<li>In a flask, prepare 200 mL of the transformation buffer containing a final concentration of 40% PEG3350, 10% DMSO, and 0.1M LiOAc. Prepare the transformation buffer immediately before use and mix it thoroughly by shaking.</li>
	<li>Remove 96-well plates from the 30 &#176;C incubator and mix them for 30 s at 1,000 rpm using the plate vortexer. Add 125 &#181;L of the transformation buffer to each well and then vortex the plates for 1 min at 1,000 rpm.</li>
	<li>Incubate the plates at 30 &#176;C for 30 min and then heat shock the yeast by placing the plates in a 42 &#176;C incubator for 15 min. 
	NOTE: Do not stack the plates.</li>
	<li>Centrifuge the plates for 5 min at 3,000 x g. 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 the top of the plates.</li>
	<li>Rinse the cells by adding to the plates 200 &#181;L of minimal dropout medium corresponding to the selectable marker on the library plasmid. 
	NOTE: We used a synthetic Ura- medium.</li>
	<li>Centrifuge the plates for 5 min at 3,000 x g and remove the supernatant over a waste bin as in step 2.10.</li>
	<li>Add 160 &#181;L of minimal Ura-medium containing 2% glucose. Vortex the plates for 1 min at 1,000 rpm and incubate them at 30 &#176;C for 48 h without shaking. After 48 h, note that a pellet of transformed yeast is visible at the bottom of each well.</li>
	<li>Using a liquid dispenser, add 100 &#181;L of Ura-media containing glucose into each well of a new set of 96-well plates.</li>
	<li>Vortex the plates containing the transformed yeast (from step 2.13) at 1,000 rpm for 30 s. 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 &#176;C for 24 h. 
	NOTE: If working with 10 plates, the medium can also be dispensed manually using multi-channel pipettes.</li>
	<li>After 24 h, a pellet of yeast should be visible in the bottom of each well containing successfully transformed yeast. To save these yeast strains as glycerol stocks, add 50 &#181;L of 50% glycerol to each well, vortex the plates for 30 s at 1,000 rpm, seal the plates with sealing tape and freeze them at -80 &#176;C. 
	NOTE: The above steps only need to be performed once. Future screenings of different yeast models against the same library disposable reagent reservoir can start from the glycerol stocks and proceed immediately from the steps below.</li>
</ol>
3. Mating Between Cells Containing Library Genes and Query Yeast
<ol>
	<li>To revive the library strains from the glycerol stock, take the 96-well plates out of the -80 &#176;C freezer, remove the sealing tape, and let the yeast thaw at room temperature for approximately 30 min.</li>
	<li>Once the yeast has thawed, use a sterile plastic 96-pin replicator to inoculate the glycerol stocks to 160 &#181;L of fresh Ura-media containing 2% glucose in 96-well plates and incubate them at 30 &#176;C for 24 h. Immediately after the library strains are used, seal the plates with sealing tape, and return them to the -80 &#176;C freezer.</li>
	<li>On the same day the library strains (W303&#945;) are thawed, inoculate the query yeast strain (here, FUS in W303a) to 50 mL of YPD and grow it overnight at 30 &#176;C with shaking at 250 rpm.</li>
	<li>The next morning, pour the query yeast strain to a sterile disposable reagent reservoir, and aliquot 160 &#181;L of the query strain to each well of a 96-well plate using a multichannel pipette.</li>
	<li>Using the liquid dispenser, dispense 160 &#181;L of YPD media into each well of ten 96-well plates. Use these plates later for mating the query yeast strain with the library yeast.</li>
	<li>Briefly vortex the 96-well plate containing the query strain and then use a sterile 96-pin replicator to transfer the query strain to the YPD plates.</li>
	<li>Briefly vortex the library strain plates and, for each library strain plate, use a new sterile 96-pin replicator to transfer the library strains to the YPD plates that have been inoculated with the query strain. 
	NOTE: Cells in glycerol lose their viability after multiple thaw-freeze cycles. Returning the library strains to the long-term storage (-80 &#176;C freezer) as soon as possible will keep the library in good quality, and ready for future use. Properly maintained, the glycerol stock can be frozen and thawed at least 10 times. We also strongly recommend keeping a working copy and backup copies of the glycerol stock. When the working stock does not work, immediately make a new working copy from the backup copy.</li>
	<li>Incubate the YPD plates at 30 &#176;C for 24 h. Note that a pellet of yeast will be visible in the bottom of each well after 24 h.</li>
	<li>Fill 96-well plates with a minimal dropout medium containing 2% raffinose, corresponding to the selectable markers on the plasmid in the query strain as well as on the library plasmid (e.g., here Ura-His-).</li>
	<li>Use a sterile 96-pin replicator to transfer yeast from the YPD mating cultures to the selective media. Incubate the 96-well plates at 30 &#176;C for 48 h; 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 media. After 2 days, observe that a pellet is visible on the bottom of the wells.</li>
</ol>
4. Spotting Assay
<ol>
	<li>After 48 h of growth in the raffinose-containing selective media, spot the yeast on agar plates.
	<ol>
		<li>Prepare 2 sets of Ura-His- dropout plates containing 2% agar using clear polystyrene plates, one containing 2% galactose and the other containing 2% glucose.</li>
		<li>Vortex the 96-well plates for 1 min at 1,000 rpm, then spot the yeast to the Ura-His- dropout plates containing 2% galactose and 2% agar (FUS and library genes are induced) and to the Ura-His- dropout plates containing 2% glucose and 2% agar (FUS and library genes repressed) using a robotic spotting machine, by which the culture in each well is spotted onto the agar plates in quadruplicate (i.e., the culture in 1 well is spotted to 4 spots on the agar plate).</li>
		<li>After spotting, let the agar plates dry, and then place them upside down in a 30 &#176;C incubator. Photograph the agar plates every 24 h to record a faster/more yeast growth in which the toxicity to the query strain is rescued or to record a slower/less growth in which the toxicity to the query strain is exacerbated. Incubate the plates for 4 days. 
		NOTE: The culture in each well can be spotted on agar plates 1-to-1, as shown in a previous transformation-based method10. However, the 1-to-4 spotting here (which can be conveniently set up using the robotic spotting machine) significantly increases the robustness of the assay by reducing the number of false positives. Positive hits are only considered when all 4 colonies from the same well show a similar phenotype.</li>
	</ol>
	</li>
</ol>

<ol><li>Dujon, B. A., Louis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+diversity+and+evolution+in+the+budding+yeasts+%28Saccharomycotina%29%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Genome diversity and evolution in the budding yeasts (Saccharomycotina).</a> Genetics. 206 (2), 717-750 (2017).</li>
<li>Khurana, V., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modelling+neurodegeneration+in+Saccharomyces+cerevisiae%3A+why+cook+with+baker%27s+yeast%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker's yeast.</a> Nature Reviews Neuroscience. 11 (6), 436-449 (2010).</li>
<li>Willingham, S., Outeiro, T. F., DeVit, M. J., Lindquist, S. L., Muchowski, P. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/14657499">Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein.</a> Science. 302 (5651), 1769-1772 (2003).</li>
<li>Outeiro, T. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yeast+cells+provide+insight+into+alpha-synuclein+biology+and+pathobiology%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Yeast cells provide insight into alpha-synuclein biology and pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li>
<li>Cooper, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson%27s+models%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313 (5785), 324-328 (2006).</li>
<li>Treusch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+links+between+Abeta+toxicity%2C+endocytic+trafficking%2C+and+Alzheimer%27s+disease+risk+factors+in+yeast%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Functional links between Abeta toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast.</a> Science. 334 (6060), 1241-1245 (2011).</li>
<li>Ju, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+yeast+model+of+FUS%2FTLS-dependent+cytotoxicity%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biology. 9 (4), 1001052(2011).</li>
<li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+determinants+and+genetic+modifiers+of+aggregation+and+toxicity+for+the+ALS+disease+protein+FUS%2FTLS%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.</a> PLoS Biology. 9 (4), 1000614(2011).</li>
<li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+yeast+TDP-43+proteinopathy+model%3A+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (17), 6439-6444 (2008).</li>
<li>Fleming, M. S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+yeast+plasmid+overexpression+screen%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">High-throughput yeast plasmid overexpression screen.</a> Journal of Visualized Experiments. (53), e2836(2011).</li>
<li>Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Life+cycle+of+the+budding+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Microbiological+Reviews%22%5BJournal%5D)">Life cycle of the budding yeast Saccharomyces cerevisiae.</a> Microbiological Reviews. 52 (4), 536-553 (1988).</li>
<li>Suter, B., Auerbach, D., Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yeast-based+functional+genomics+and+proteomics+technologies%3A+the+first+15+years+and+beyond%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond.</a> Biotechniques. 40 (5), 625-644 (2006).</li>
<li>Barmada, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amelioration+of+toxicity+in+neuronal+models+of+amyotrophic+lateral+sclerosis+by+hUPF1%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (25), 7821-7826 (2015).</li>
<li>Jackson, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preservation+of+forelimb+function+by+UPF1+gene+therapy+in+a+rat+model+of+TDP-43-induced+motor+paralysis%5BTitle%5D)+AND+%22Gene+Therapy%22%5BJournal%5D)">Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.</a> Gene Therapy. 22 (1), 20-28 (2015).</li>
</ol>

The authors have nothing to disclose.

Here, we describe a protocol to perform a plasmid overexpression screen in yeast using mating to introduce the plasmid library into the yeast model. Using this approach, multiple yeast models of neurodegenerative disease protein toxicity can be screened using the same collection of yeast transformed with a plasmid library. The laborious process of transformation only needs to be performed once, after which highly efficient yeast mating is used to introduce the plasmid library into the query strain. This protocol does rel...

The budding yeast Saccharomyces cerevisiae has been widely used in basic scientific research1 to understand cellular processes directly related to human diseases. Moreover, it has been used as a model organism for studying human disease-associated proteins, such as those linked to the most common neurodegenerative diseases, including Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, and Amyotrophic Lateral Sclerosis (ALS)2. An advantage of the yeast model is the ease with which a genome-wide screen can be performed to identify cellular pathways related to the toxicity of disease-related proteins, thus giving insight into the mechanism of their toxicity. One such screen is called an overexpression library screen, in&#160;which each of the 5,500 yeast genes in an arrayed library is transformed into a yeast model to identify which genes can modify toxicity when overexpressed. This screening method has been successfully applied in the yeast models of multiple neurodegenerative disease-associated proteins, including huntingtin for Huntington&#39;s disease3, &#945;-synuclein for Parkinson&#39;s disease4,5, A&#946; for Alzheimer&#39;s disease6, and FUS and TDP-43 for ALS7,8,9. While it is usually done in a high-throughput manner10, the most labor-intensive step of the screen is individually transforming 5,500 yeast genes from an arrayed library. This step must be performed each time the screening is repeated, and whenever a newly established yeast model needs to be studied. It is important to find a more efficient way to accomplish this task.
Yeast cells can stably exist in both haploid and diploid forms. There are two opposite mating types of haploid cells, mating type a and &#945;. Haploid cells of each mating type produce and secrete their own specific mating pheromone, to which only the opposite mating type cells respond. This allows mating between a and &#945; cells to produce stable diploid cells, a/&#945;. This process is spontaneous and highly efficient11. We can take advantage of this unique life cycle of S. cerevisiae to introduce the plasmid library. More specifically, each gene in the arrayed plasmid library will be transformed into haploid cells of one mating type, i.e., &#945; cell. These cells containing the library genes will then be stored in glycerol stock in an arrayed 96-well format. For each yeast model that needs to be screened, yeast cells containing the library genes can be thawed from the glycerol stock, and the screening can be done through&#160;mating with the yeast model of interest in the opposite mating type, i.e., mating type a. This idea of using mating to bring together two genes into yeast is not new. It has been successfully applied in the high-throughput yeast two-hybrid screening, in which a bait construct (i.e., Gal4-DNA-binding domain fusions) in one mating type is brought together through mating with a prey construct from an arrayed library12. However, this strategy has never been applied in&#160;overexpression library screenings, which have always used traditional transformation methods.
Our laboratory previously established a yeast model of the ALS-associated protein FUS7. Through overexpression library screening using the transformation method we discovered&#160;five yeast genes (ECM32, NAM8, SBP1, SKO1, and VHR1) that rescue toxicity of FUS when overexpressed. These findings were independently confirmed with a similar study by another group8. hUPF1, a human homolog of ECM32, was later shown to suppress toxicity in primary neuronal cells13&#160;and in an animal model of ALS14 as well. Using these five genes as proof of principle, we demonstrate that all five genes similarly rescue FUS toxicity when they are introduced into the FUS yeast model by mating. Since yeast cells containing the library genes can be stored permanently in glycerol stock and revived whenever needed, this mating-based method will remove the time-consuming step of transformation each time the library needs to be screened against. Since mating is highly efficient with no plasmid transformation involved, this strategy also significantly decreases the cost associated with purification and transformation of a large plasmid library. We will successfully apply this method to a library screening against yeast model of FUS.
The procedure for mating-based screening is briefly described in Figure 1. Initially, the arrayed plasmid library is transformed into a haploid yeast strain of mating type &#945; using a high-throughput yeast transformation protocol in which each well of a 96-well plate contains yeast transformed with a specific library plasmid. This collection of transformed yeast is saved as a glycerol stock that can be thawed and revived for use later on. The yeast model of interest, in&#160;this case FUS toxicity, must be generated in a haploid yeast strain with the opposite mating type (mating type a). In a high-throughput manner using sterile 96-pin replicators, the FUS strain and yeast strains containing the plasmid library are transferred to 96-well plates containing rich media and allowed to mate. Following&#160;mating, a small volume from each well of the mating culture is transferred to 96-well plates containing synthetic dropout media in which only diploid yeast containing both the FUS and library genes can grow. A robotic spotting machine is then used to transfer yeast culture from each well onto agar plates where the expression of FUS and the library genes is induced.&#160; Additionally, yeast culture is spotted&#160;to control agar plates where FUS and the library genes are not expressed. Following&#160;growth on&#160;agar plates, genes that rescue or exacerbate FUS toxicity will be identified.

<table><tbody><tr><td>salmon Sperm DNA (SS-DNA)</td><td>Sigma-Aldrich</td><td>&amp;nbsp; D1626</td><td></td></tr><tr><td>YPD broth</td><td>Research Products International (RPI)</td><td>Y20090</td><td></td></tr><tr><td>Granulated Agar</td><td>Fisher Sci</td><td>BP97445</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Research Products International (RPI)</td><td>G32040</td><td></td></tr><tr><td>D-(+)-Galactose</td><td>Research Products International (RPI)</td><td>G33000</td><td></td></tr><tr><td>D-(+)-Raffinose Pentahydrate</td><td>Research Products International (RPI)</td><td>R20500</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Fisher Sci</td><td>A702-500</td><td></td></tr><tr><td>Synthetic Ura- drop out medium</td><td>Clontech</td><td>630416</td><td></td></tr><tr><td>Yeast amino acid drop out supplement -Histidine/-Uracil</td><td>Clontech</td><td>630422</td><td></td></tr><tr><td>Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate</td><td>Research Products International (RPI)</td><td>Y20060</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Sci</td><td>S67496</td><td></td></tr><tr><td>Lithium acetate, anhydrous</td><td>Fisher Sci</td><td>AC268640010</td><td></td></tr><tr><td>Polyethylene Glycol 3350 (PEG-3350)</td><td>Spectrum Chemical&amp;nbsp;</td><td>PO125-12KG</td><td></td></tr><tr><td>96 Pin Replicator&amp;nbsp;</td><td>Scinomix</td><td>SCI-5010-OS</td><td></td></tr><tr><td>Nunc OmniTray</td><td>Thermo Sci</td><td>140156</td><td></td></tr><tr><td>Corning Costar 96 well assay plate, round bottom with lid</td><td>Fisher Sci</td><td>07-200-760</td><td>non-treated, sterile</td></tr><tr><td>Eppendorf&amp;nbsp;Research plus Multichannel Pipette</td><td>Eppendorf</td><td>TI13690052</td><td>30-300ul volume</td></tr><tr><td>Fisherbrand&amp;nbsp;Isotemp Digital Dry Baths/Block Heaters</td><td>Fisher Sci</td><td>88-860-023</td><td></td></tr><tr><td>Eppendorf&amp;nbsp;MixMate</td><td>Eppendorf</td><td>21-379-00</td><td></td></tr><tr><td>Eppendorf 5810R Centrifuge</td><td>Fisher Sci</td><td>05-413-112</td><td></td></tr><tr><td>Avanti J-26 XPI Centrifuge</td><td>Beckman</td><td>393127</td><td></td></tr><tr><td>MultiFlo FX Multi-Mode Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Rotor HDA&amp;nbsp;&amp;nbsp;</td><td>Singer Instruments</td><td></td><td></td></tr></tbody></table>

mating-based overexpression library screening in yeast

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

The ALS-associated protein FUS, an RNA/DNA binding protein, was previously studied in haploid yeast7,8. Genetic screening using the transformation-based method discovered several yeast genes that suppress FUS toxicity. The human homolog of one of the yeast genes was later demonstrated to be effective at suppressing toxicity in a primary neuronal cell and rat model of ALS13. Here, we are using the same yeast...

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

mating-based-overexpression-library-screening-in-yeast

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This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Protein Engineering by Yeast Surface Display

Learn a mating-based overexpression screening method in budding yeast using an arrayed plasmid library. This technique efficiently identifies genetic factors that regulate toxicity of neurodegenerative disease proteins.

Mating-based Overexpression Library Screening in Yeast

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