The genetics of S. cerevisiae

Biology 2

Genetics of Saccharomyces cerevisiae
Part 1: Introduction & Determination of Phenotypes
Overview
You will be given three strains of Saccharomyces cerevisiae (named A, B, and C), each with a different genotype. The hypotheses for this study are (1) identify the phenotypes and genotypes of the three strains in regards to their ability to synthesize leucine, and uracil; and
(2) determine whether these genes follow Mendel’s Second Law and assort independently.
Goals and objectives of this project:
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•
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Learn how to work with S. cerevisiae, a model organism that is powerful for genetic research Experimentally explore genetic concepts (auxotroph vs. prototroph, genes vs. alleles, diploid vs. haploid, complementation, segregation and independent assortment, meiosis vs. mitosis, genotype vs. phenotype)
Develop and test scientific hypotheses
Learn to work with a microscope, micropipettor, microcentrifuge and hemacytometer
Learn about the genetics of metabolism

How this project will be graded:
1. Your scores for Question Sets 1, 2, and 3 will be combined into a single grade. That grade will be included in your Lab Average for the course. Your Lab Average will be included in your final semester average as described in the syllabus for this course. (the deadline for the question submission will be determined in class depending on the progress of the lab)
2. Your scores for Question Sets 4, 5, and 6 will be combined into a single grade. That grade will be included in your Lab Average for the course. (the deadline for the question submission will be determined in class depending on the progress of the lab)
3. You will write a formal lab report in a style and format suitable for submission to the journal Molecular & Cellular Biology. Your report will be included in your final semester average as described in the syllabus for this course.

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Introduction
For background on yeast, read the Yeast Wiki.
Yeast can be grown in the laboratory in two types of media:
1. YPD (yeast peptone dextrose): which contains all necessary nutrients so the yeast do not need to synthesize their own. Auxotrophic yeast strains, which are unable to synthesize all of their own nutrients, will grow on YPD.
2. SD (synthetic dextrose): which does not contain all nutrients so the yeast need to synthesize their own. Specific metabolites (like the amino acids or nitrogenous bases) can be added to the media, when necessary. Prototrophic yeast strains, which can synthesize all of their own nutrients, will grow on plain SD plates (without any nutrients added). In the laboratory, yeast cells grow most quickly in liquid YPD media, with a doubling time of
90 minutes at 30°C. This liquid culture is placed into a flask or a tube. The container is constantly shaken, to ensure that the yeast do not settle on the bottom and that all cells are equally exposed to atmospheric oxygen. On agar-containing solid plates (which can be YPD or SD), an individual yeast cell forms a visible colony in two or three days.
Question Set 1
Answer these questions completely:
1. What are four reasons that S. cerevisiae is a great model organism for research?
2. What is the difference between asexual and sexual reproduction in yeast?
3. How do yeast convert from a haploid to a diploid?
4. How do yeast convert from a diploid to a haploid?
5. Proteins are chains of amino acids. There are twenty different types of naturally occurring amino acids, and all organisms must have all twenty of them in order to survive. In this project we will be discussing the amino acid leucine. What are the two possible methods that a cell may employ to be certain that it has each of the 20 amino acids? 6. RNA is partly made of the base uracil. What are the two possible methods that a cell may employ to be certain that it has uracil?
7. Suppose that you have a particular S. cerevisiae mutant strain that cannot make the amino acid leucine. Will the strain grow on a YPD plate? What about a SD plate?
How could you modify the SD plate to allow this strain to grow?

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Growing yeast
You will be given 3 strains (named A through C), each with a different genotype. The ultimate goal is to study the genetics of these strains and determine how the strains are different. The first step is to grow these strains, so that subsequent steps can be carried out.
Use sterile technique! Before you begin, wipe your bench down with water so that it is free of dust. Keep the lids on the plates at all times, except for the few seconds it takes to work with the yeast. Do not touch any sterile surface with your hands or other object. Both the toothpicks and inside of the plates are sterile.

1. Label a fresh YPD plate as shown in the figure to the right. Label the side of the plate containing the media (NOT the lid). The covered plate will be grown upside-down (with the lid facing down). Do not draw the squares; that is where the yeast will go. 2. Using a toothpick, gently pick up part of a colony of the correct strain from the instructor’s plate. Do not penetrate the agar-based YPD media, but just take the yeast from the top.
You need very little amount of yeast, so they will be barely visible on the toothpick.
3. Use several gentle streaks to spread the strain in a “patch” (indicated by the square) in the correct location on the labeled YPD plate. Again, do not penetrate the agar-based
YPD media, but place the yeast gently on the top. And there will be very little amount of cells, so they will be barely visible on the plate.
4. Repeat for the other strains.
5. When finished with all the strains, place the covered plate (with the lid facing down) in the
30°C incubator and grow for 24 hours. If you don’t proceed to the next step immediately after the 24 hours, place the plate at 4°C.
Question Set 2
1. How do the patches grow from very few cells (what biological process is used)?
2. In terms of genotype, how are all the resulting cells in a single patch related to each other? 3. With this YPD plate, can you determine whether patch A, B and C are different genotypes? Explain.

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Replica plating To test the phenotypes of the strains that you grew, you will create a “copy” of the YPD plate onto selective plates. In the section following this one, you will monitor whether the strains grow on the selective plates, and record each strain’s phenotype. From the phenotypes, you can make a prediction of the genotypes.
Important note: All plates will contain histidine; however this phenotype will not be analyzed in this project

Label all the plates with your initials, date and media type (four plates: SD+His, SD+His+Leu,
SD+His+Ura, SD+His+Leu+Ura). Label the side of the plate containing the media (NOT the lid). 1. Place the replica block without the ring on the lab bench. Make sure there is a line on one width of the block ring, to allow plate alignment. Place a sterile velvet in the center of the block (velvet side up). Only touch the sides of the velvet, to maintain a sterile center. Put a ring down over the velvet, making sure not to touch the center of the velvet. The line on the ring should be facing away from you. Make sure the velvet is pulled down tightly and evenly by the ring.
2. With a marker, make a short line at the top of the original YPD plate (on the side containing the media). Remove the lid from the original YPD plate. Turn the plate upsidedown and align the line on the plate to the line on the ring. Gently press the media and colonies down onto the velvet, to leave a “copy” of the yeast on the velvet. Make sure yeast are evenly transferred by gently pushing down 4 times in different parts of the plate.
Remove the plate from the block and replace the lid of the plate.
3. Transfer a copy of the yeast to several types of plates, as follows. First, make sure there is a short line at the top of each plate. Then, remove the lid from the labeled SD+His plate.
Turn the plate upside-down and align the line on the plate to the line on the ring. Gently press the media of the plate down onto the velvet, as you did above. Remove the plate from the block and replace the lid of the plate.
4. Using the same velvet that contains a copy of your strains, perform the same procedure for the following plates: SD+His+Leu, SD+His+Ura, and SD+His+Leu+Ura.
5. Discard the velvet into a tub of soapy water.
6. NOTE: Many cells from a patch can sometimes be copied to a new plate, making it look like that patch is growing on the new plate when the patch actually is not.
Immediately after replica plating, make a blue mark on the new plate where there looks like a lot of growth. When it comes to recording growth of these colonies, a
“+” means that there is further growth.
7. Once all of the replica plating is finished, place the covered plates (with the lid facing down) in the 30°C incubator. Include the original YPD plate. In the next section, you will observe the plates after 24 and 48 hours of growth. Page 4 of 21

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Observing phenotypes 1. After 24 hours, record the growth of each strain on each plate. Place + in the appropriate box for growth and – for no growth.
Table 1. Growth of S. cerevisiae strains on different types of plate media.
Strain
A
B
C

SD+His

SD+His+Ura SD+His+Leu

SD+His
+Ura+Leu

2. Put the plates back into the incubator and grow for another 24 hours. Record growth by adding + or – for each box after the previous marking. For example, if a strain did not show growth after the first 24 hours, but did show growth after the next 24 hours, the box should contain -+.
3. Using the data above, fill in the following table. Write the phenotype of each strain: for example, Ura- Leu+ is a strain that cannot synthesize uracil but can synthesize leucine.
Write whether each strain is a prototroph or auxotroph.
Table 2. Phenotype summaries of selected S. cerevisiae strains.
Strain

Phenotype

Prototroph or auxotroph

A
B
C

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Question Set 3
1. Please provide a copy of Table 1 and Table 2.
2. These three strains are haploids and have the following mating types: A = MATa, B =
MATa, and C = MATalpha. Based on the data that you have up to this point in time, what is the most likely genotype for each of those three strains?
3. What is the purpose behind replica plating onto SD+His+Leu+Ura plates in this experiment? 4. How many different auxotrophic phenotypes are represented in your collection of three yeast strains?
5. Suppose you had a fourth yeast strain that was haploid and unable to synthesize the amino acid, arginine. If you replica plated the strain onto the same kinds of plates that you used in this experiment, on which plates would this strain be able to grow? Explain your answer. Page 6 of 21

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Genetics of Saccharomyces cerevisae
Part 2: Mating & Observations of Diploids
Question Set 4
1. To determine the cause of the auxotrophy in two of your yeast strains, you are going to set up a cross, B x C. Why can these particular strains mate with each other?
2. A diploid will be formed from the B x C cross. What is the predicted genotype of this diploid? 3. On which plates should the diploid grow (SD+His, SD+His+Leu, SD+His+Ura,
SD+His+Leu+Ura)?
4. Which plates allow the diploid to grow, but not the two haploid parents? (The word
“complementation” must be in your answer.)
5. What do you think will be the genotype of the mating type locus in this diploid?

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Crossing strains
In order to study the genetic basis of the Leu- auxotrophies, you are going to perform a cross,
B x C.

1. Label a fresh YPD plate as shown in the figure to the right. Label the side containing the media (NOT the lid). The covered plate will be grown upside-down
(with the facing down). Don’t draw the square; that is where the yeast will go.
2. Using a toothpick, gently pick up a small streak of the B patch from your original
YPD plate.
3. Use several gentle streaks to spread the strain in the patch on the new labeled
YPD plate.
4. Then, using a new toothpick, do the same for the C strain, and extensively but gently mix the B and C cells together in the same patch. This will increase the direct contact of
MATa cells and MATalpha cells.

5. Once the mating plate has been created, place the covered plate (with the lid facing down) in the 30°C incubator. Grow for 24 hours, which will allow mating and the subsequent formation of diploid cells.

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Selection of diploids
After 24 hours, the cross patch will consist of two types of cells: haploids that did not mate, and diploids. You will now test whether these cells grow on an SD+His plate.

1. Label a fresh SD+His plate as shown in the figure to the right. Label the side containing the media
(NOT the lid). As usual, the covered plate will be grown upside-down (with the lid facing down).
2. Use a toothpick to collect a small amount (barely visible) of the “parent” B strain from the original
YPD plate.
3. You will streak “for single colonies”, using proper microbiological technique as in the figure below (from http://molecular-biology-protocols.readthedocs.org/en/latest/E.coli.html). The difference is that you will be doing this in one sector (labeled “B”) of the SD+His plate.
This technique causes a reduction in cell concentration with each subsequent set of streaks, until you ideally are spreading single cells across the plate.
4. To do this, make four streaks of the cells collected above along the edge of the B sector. This should take up ¼ of the sector. Then use the other side of the toothpick (which has no cells) to make 4 additional streaks at at angle (as shown in the figure). This should take up another ¼ of the sector. Next, use a new toothpick to create streaks in the 3rd quarter. And then use the other side of this toothpick to continue the fourth quarter. 5. Repeat this technique for the C strain.
Then repeat this for the B x C mating mixture from the YPD mating plate.
6. Once this plate has been completed, place the covered plate (with the lid facing down) in the 30°C incubator.

7. Observe growth every 24 hours, as described in the next section.

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Observation of diploids
1. Using the following table, record growth on the SD+His plate after 24, 48 and 72 hours of growth. Use the + and – symbols as above (for example, -++ means that the strain did not grow after 24 hours, but did grow after 48 and 72 hours).
Table 3. Testing strains for complementation.
Strain
B
C
BxC

Growth on SD+His

2. After observing the plates at 72 hours, put the plates at 4°C until the next step.
Question Set 5
1. Please provide a copy of Table 3.
2. Looking back at Question #4 from Set 4, do your results in Table 3 support your predictions? Page 10 of 21

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Genetics of Saccharomyces cerevisae
Part 3: Sporulation & Observations of Haploids

In the laboratory, a diploid cell can be promoted to undergo sporulation (meiosis), a process that results in four haploid spores. In this section, you will promote sporulation by growing the diploid cells in a 1% potassium acetate solution.
1. Using tape and a marker, label a test tube with your initials, date and “SPO” (sporulation).
2. Using a micropipet, add 0.5 ml of 1% potassium acetate to the test tube.
3. Using an applicator stick, pick up one diploid colony (from the B x C cross) off the SD+His plate. Shake the cells off the stick and into the 0.5 ml within the test tube. Vortex the tube briefly. 4. Place the tube on the roller drum at room temperature (which is optimal for sporulation).
Grow for at least 4 days (to allow time for sporulation to occur).

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Question Set 6
1. Assuming that the LEU, URA, and MAT loci assort independently, predict the phenotypic/genotypic ratios that you expect for the spores. The table on this page is for your scratchwork only! Be sure that the table you submit to me is in ASM format (in other words, suitable for publication in the journal Molecular & Cellular Biology).
Genotype

Phenotype

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Proportion of spores

The genetics of S. cerevisiae

Biology 2

Monitor sporulation and measure cell concentration by microscopy
In this section, you will examine the cells under the microscope to observe whether sporulation has occurred. Also, you will measure the cell concentration using a hemacytometer. 1. Prepare a hemacytometer and coverslip by making sure they are clean and dry. If not, clean them with a 70% ethanol solution. This solution will dry rapidly as the ethanol evaporates. 2. Once both components are dry, place the coverslip onto the center of the hemacytometer.
3. Using a micropipet, slowly place 80% should be tetrads. The rest are unsporulated diploids. 7. NOTE: Your cell concentration may be too high to count. In this case, on the hemacytometer, the entire surface will be covered with cells. Dilute the cells by taking
100 µl of the original sporulation mixture and adding to 900 µl of water in a fresh microcentrifuge tube. Close the cap and mix by inverting several times. Then, place 100 total cells.
10. Once you have counted cells, you need to determine the cell concentration of the solution.
To do this, use the following formula: C = n * 25 * 104 / b, where C = cell concentration
(cells per ml), n = number of cells counted, and b = number of blocks counted.
11. Each member of the group should count >100 cells and then determine the resulting cell concentration. 12. Compare the values obtained by each group member. Use the average of the group as your actual concentration.

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Plate the diploids and haploids (spores)
The medium contains a mixture of unsporulated diploids and tetrads. You will digest the cell wall of the tetrad with zymolyase, releasing the spores. Then you will dilute this cell suspension so that you can plate a certain number of cells on a YPD plate.
1. Using a micropipet, take 200 µl of cells from the test tube and place into a fresh microcentrifuge tube labeled with your initials and “A”.
2. Centrifuge the tube for 15 seconds at maximum speed, using a microcentrifuge.
3. The pellet contains the cells. Remove the supernatant with a micropipet, being careful to not disturb the pelleted cells.
4. Using a micropipet, add 50 µl of zymolyase (an enzyme that digests the ascus, releasing the spores inside) to the cell pellet.
5. Resuspend the cells in the pellet by pipeting the solution up and down, until the pellet has been totally disrupted.
6. Place the tube in a 30°C water bath for 30 minutes, to allow the zymolyase to act.
7. Add 950 µl of dH2O to the tube. Pipet up and down slowly, in order to break apart the tetrads and free the individual spores.
8. The cells have now been diluted 5-fold. The cells were originally in 200 µl but now are in
1ml, or 1000 µl. Using your previously determined cell concentration, calculate the cell concentration in the current suspension.
9. Add 10 µl of this cell suspension into another eppendorf tube (labeled B) with 990 µl of dH2O. Close the cap. Mix by inverting up and down 10 times. This is a 100-fold dilution.
Calculate the cell concentration in this cell suspension (B).
10. Do this 100-fold dilution again: add 10 µl of the cell suspension from the previous step
(tube B) into another eppendorf tube (labeled C) with 990 µl of dH2O. Close the cap. Mix by inverting up and down 10 times. Calculate the cell concentration in this cell suspension (C).
11. You want to create a solution at 1000 cells per ml. Choose whether B or C is closer (yet above) 1000 cells per ml. You will use this tube to perform a further dilution into yet another eppendorf tube, labeled D.
12. Calculate the further dilution using C1V1 = C2V2. C1 = the cell concentration you calculated for B or C. V1 = the volume (in ml) from tube B or C (unknown volume that you are trying to calculate), C2 = 1000 cells/ml (final concentration), V2 = 1 ml (final volume).
13. From this formula, you will calculate the volume that you need from tube B or C (V1) that you will add to tube D. You need to bring the final volume of tube D up to 1 ml. For
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example, if you calculate V1 = 0.1 ml, then the amount of water to add to tube D is 1 ml –
0.1 ml = 0.9 ml. Once the water and cells are added, close the cap and invert 10 times to mix. 14. Label a YPD plate with “sporulated”, your initials and the date.
15. Using a micropipet, 100 µl of the cell suspension to the YPD plate. Use a sterile spreader to spread the liquid evenly around the plate until dry. If still wet after spreading for a minute, leave the covered plate with the lid facing upwards until the liquid has dried
(overnight if necessary).
16. Place the plate in the 30°C incubator, and leave for exactly 48 hours (if you dry the plates overnight, that counts towards the 48 hours).
17. After exactly 48 hours, place the plates at 4°C until ready for the next step.

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Question Set 7
1. Calculate how many cells were placed onto the YPD plate in the last section. Show your work.
2. On the YPD plate, a colony could be either a haploid spore or an unsporulated diploid.
Importantly, we are only interested in the haploids when determining the genotypic and phenotypic ratios because the haploids have gone through meiosis. How could you determine which are haploids? (Hint: diploids cannot mate.)
3. Below, you will perform a mating test to determine whether a colony is MATa or
MATalpha. Which specific result will tell you that a colony is MATa?

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Count the colonies and replica plate
After 48 hours, colonies will form on the YPD plate. You will replica plate these colonies to plates with selective media to test for phenotypes.
There are two types of plates you will use to test the mating type of each colony: a MATa lawn and a MATalpha lawn. Each plate is covered with a “lawn” of MATa or MATalpha cells
(and the cells are specifically designed for this mating test). Your YPD plate containing the colonies will be replica plated to MATa and MATalpha tester lawn. Then, the tester plates
(with your colonies and the lawn) are grown for 24 hours at 30°C, to allow mating to occur.
After this, the cells on the mating tester plate are replica plated to an SD plate. . If your colony is of the opposite mating type as the tester strain, a diploid will form which is able to grow on an SD plate.
1. Count the number of colonies on the plate. Count while the plate is upside-down (lid down) and number the colony by writing on the plastic (small but clear: 1, 2, 3, etc) to indicate that you have counted that colony and to later identify the colony.
2. You are going to transfer a copy of the YPD plate to several types of plates, as you did above. Prepare the original YPD plate and the following plates with alignment lines as you did earlier: SD+His, SD+His+Leu, SD+His+Ura, SD+His+Leu+Ura, MATa lawn,
MATalpha lawn.
3. Replica plate as you did earlier, making sure to use the alignment lines. When copying cells from the velvet to fresh plates, follow the order of plates in step 2, but stop after transferring cells to the MATa lawn. The velvet now has been contaminated with your colonies and the MATa lawn, and you don’t want the MATa lawn on subsequent plates.
Discard the velvet into soapy water.
4. Obtain a fresh velvet and then make another copy of the original YPD plate onto this velvet. Then copy the colonies from this velvet to the MATalpha lawn. Discard the velvet into soapy water.
5. NOTE: Many cells from a colony can sometimes be copied to a new plate, making it look like that colony is growing on the new plate when the colony actually is not.
Immediately after replica plating, make a blue mark on the new plate where there looks like a lot of growth. When it comes to recording growth of these colonies, a
“+” means that there is further growth.
6. Once all the replica plating is finished, place the covered plates (with the lid facing down) in the 30°C incubator. Include the original YPD plate.

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Obesrve phenotypes of the haploids
1. Grow the replica plates at 30°C. Observe the plates every 24 hours for 72 hours. After the first 24 hours, match colony# on all the plates, by accurately superimposing each replicated plate onto the original YPD plate. All colonies should line up perfectly between the replicated plate and the original YPD plate. Once you have lined up the plates, copy all of the colony #’s to the replicated plates.
2. After each 24 hours, use the table on the next page to fill in the phenotypes for each haploid (using the colony# from the original YPD plate). Place “+” in the appropriate box for growth and “–“ for no growth. For example, if a strain did not show growth after the first 24 hours, but did show growth at 48 and 72 hours, the box should contain -++.
3. To test mating type, you need to replica plate each mating lawn to an SD plate. Grow the two SD plates for 24-48 hours at 30°C.
Observe mating type
1. Observe the SD plates for growth at 24 and 48h. Match each colony with the correct colony# as above. Place a “+” in the MATa column if there was growth on the SD plate after mating with the MATa testing lawn and a “-“ if there was no growth. Likewise, place a “+” in the MATalpha column if there was growth on the SD plate after mating with the
MATa testing lawn and a “-“if there was no growth.

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Colony#

SD+His

SD+His+Ura SD+His+Leu

Determining genotypes and testing predictions
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SD+His+
Ura+Leu

MATa lawn MATalpha lawn The genetics of S. cerevisiae

Biology 2

1. Earlier, you predicted the genotypic ratios that you expect for the haploid spores. Here, list those “genotypes” in the first column.
2. Then fill in the “number observed” column using the data from the previous table.
3. Use the ratios that you predicted earlier to determine the “number expected” in this experiment. (Remember chi-square? Multiply each ratio by the total “observed” number to get the “expected” number.)
4. Test whether your results matched your predictions by completing the chi square analysis.

Genotype

Number observed Number expected TOTAL

••• PLEASE NOTE that the tables on this page and the preceding page are for recordkeeping and scratchwork purposes only.

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