Heat Shock Proteins in Yeast (2012)

Transcription

1 MASSACHUSETTS INSTITUTE OF Technology Department of Biology Heat Shock Proteins in Yeast (2012) Summary Lydia Breen (Stoneham High School) Mary Brunson (Brookline High School) Yeast is a single-celled eukaryote in the Kingdom Fungi. They are a very diverse group of microorganisms that are useful in such processes as bread and alcohol production. Many yeast species have been found to survive best at 30 C, although there are some species that can survive at other temperatures. Yeast are an important model organism in the biology lab; scientists have used yeast to discover many things about how human cells function. In this activity, student test the ability of typical baker s yeast to survive in different temperatures and see the heat shock response when yeast are first placed in a slightly warm environment before entering the very warm environment of 51 C. This activity is designed for an upper-level biology course, but could be adapted for other levels by adding or removing certain concepts (described in Extensions below). It should be noted that this procedure has not been tested as written. Key Concepts Massachusetts Curriculum Frameworks: SIS1. Make observations, raise questions, and formulate hypotheses. SIS2. Design and conduct scientific investigations. SIS3. Analyze and interpret results of scientific investigations. 1.2 Describe the basic molecular structures and primary functions of the four major categories of organic molecules (carbohydrates, lipids, proteins, nucleic acids). AAAS benchmarks: o The work of the cell is carried out by the many different types of molecules it assembles, mostly proteins. Protein molecules are long, usually folded chains made from 20 different kinds of amino acid molecules. The function of each protein molecule depends on its specific sequence of amino acids and its shape. The shape of the chain is a consequence of attractions between its parts. 5C/H3 o Complex interactions among the different kinds of molecules in the cell cause distinct cycles of activities, such as growth and division. Cell behavior can also be affected by molecules from other parts of the organism or even other organisms. 5C/H5 o Some protein molecules assist in replicating genetic information, repairing cell structures, helping other molecules get in or out of the cell, and generally catalyzing and regulating molecular interactions. 5C/H9** (SFAA) Objectives At the end of this activity, students will be able to

2 MASSACHUSETTS INSTITUTE OF Technology Department of Biology Describe the importance of heat shock proteins in yeast that are temperature stressed. Analyze results from a controlled experiment to formulate a conclusion about yeast heat shock proteins. Materials Dry Baker s yeast YPD broth Petri dishes with YPD agar (yeast extract, Bacto-peptone, glucose, distilled water, agar) Micropipettes or transfer pipettes Test tubes Water baths or incubators (some way to keep yeast at 3 different temperatures) Procedure 1. Teacher prep before day 1: a. The following website provides information sterile techniques, making cultures, and yeast physiology: (click on yeast experiments ). b. Two or three days before you intend to do the experiment, create a plate of yeast by combining 0.1 g of baker s yeast with 10 ml of water, then streak for single colonies (to learn how to do this, watch the video at the following link: bacteria- for- a- single- colony/). Let that plate sit overnight in a 30 C incubator if possible. If you don t have access to an incubator, store at room temperature, but it may take 2 nights. You can put your plates in the refrigerator after they have grown. You may want to start this a few days in advance to make sure you get good growth. Once growth happens, you can place your plate in the refrigerator until you are ready to use it for the next step c. One day before (after your stock plate is made), select one colony and suspend it in about 3 ml of YPD broth (you can buy this, or make it yourself using the following recipe: 5g yeast extract, 10g Bacto-peptone, 10g glucose, and 500 ml distilled water). You can do this with a sterilized wooden stick or an inoculating loop. Let this liquid culture sit overnight in a 30 C incubator or at room temperature. If you have a shaker or roller wheel, you should use that to aerate your yeast culture overnight. If you do not have access to either of these, you should put it in a very large Erlenmeyer flask to give the culture a large surface area. Make sure containers are sealed to prevent contamination. This is the culture you will need on the first student day for students to use. You will also need this overnight culture to prepare the solutions for the second day of student experimentation. d. At the end of the first day of student experimentation (or the day before you want them to run the second part of the experiment), dilute your overnight culture in YPD broth by adding 50 µl of overnight to 20 ml of water (if you plan on using the culture for a morning class with this dilution, the culture was ready after sitting for hours). If you plan on using the culture for a class after noon, you should add 10 µl to 20 ml of water and let that sit overnight. You should do this step as late in the afternoon as possible so it will be ready for use in class. e. Also before the first day, make enough YPD plates for each group of 3-4 students to use 3 plates. YPD plates can be made using the following recipe: 20 g agar, 10 g yeast extract, 20 g Bacto-peptone, 860 ml distilled water. Autoclave the media, then add 100 ml of 20% sterile glucose.

3 MASSACHUSETTS INSTITUTE OF Technology Department of Biology 2. Students will follow procedure in student handout. If possible, you should use Bunsen burners or flames to keep all tubes sterile. At the very least, make sure students follow sterile techniques as often as your available materials allow. 3. After the students get their own data, you should discuss expected results and the biology behind heat shock/chaperone proteins a. The yeast that are exposed to the 51 C bath will survive better when they were first exposed to the 37 C bath for 30 minutes. b. This is because the yeast produce heat shock proteins that act as protein folding chaperones. They keep the proteins properly folded, even when the temperature is high enough to normally denature them. Extensions Have students check other temperatures (not 37 C to see if they induce production of heat shock proteins. Have students do time trials for different amounts of time in the 37 C bath before placing it in the 51 C bath (does more time prepare them more?) Have students check lower temperatures. Remove the data tables from the write-up and have them develop their own meaningful data tables. Have students set up the cultures instead of teachers. Assessment Performance Walk around and discuss the procedure and hypotheses with the students as they are doing it. Product The activity handout with observations and summary questions answered. Additional Resources yeast information - yeast information and lab techniques

4 Yeast response to heat Introduction: Yeast are single- celled eukaryotes in the Kingdom Fungi. They are a very diverse group of microorganisms that are useful in bread and alcohol production. Many yeast species have been found to survive best at 30 C, although there are some species that can survive at other temperatures. Yeast cells produce many of the same proteins that are produced in human cells. Therefore, yeast can be an important model organism in the biology lab. Scientists have used yeast to discover many things about how human cells function. In this lab, we will test the ability of typical baker s yeast to survive in different temperatures. You will make predictions about what will happen when you place the yeast in different environments for set periods of time. When doing this lab, you should remember what you learned about the biochemistry of proteins. Each protein has a distinct, specific structure that is necessary for its function. That protein structure is largely held together by hydrogen bonds. When proteins are heated, those bonds become unstable and are easily broken. An example of this is when you cook eggs. The egg white protein denatures when you heat it up, causing them to bunch together and solidify into the cooked egg white that you eat. Materials: (per group of 4) Day 1: 3 test tubes Incubator/water bath (30 C, 37 C, and 51 C) Petri dish with YPD growth media Small transfer pipet or micropipette Timer Day 2: 2 test tubes Incubator/water bath (30 C, 37 C, and 51 C) 2 Petri dishes with YPD growth media Small transfer pipet or micropipette Sterile cotton swabs (2) Timer Procedure Day 1: 1. Make predictions. What do you think will happen if yeast is exposed to the following temperatures? a. 30 C: b. 37 C: c. 51 C: 2. Label your three test tubes with your group name and 30 C, 37 C, and 51 C. 3. Take the overnight culture that your teacher gave you and distribute 300 µl into 3 separate test tubes using the micropipettes. 4. Place each test tube in the appropriate location (there are water baths and incubators set up) and set the timer for 30 minutes. After 30 minutes, remove the test tubes from their location and place in the 30 C incubator or water bath. 5. You should take a YPD plate (prepared by your teacher) and label the bottom of the plate with your group name and 3 small dots (with a Sharpie) distributed throughout the dish. See the diagram to the right as an example. 6. Mix your test tubes using a vortex or swirl them. Using the micropipette, place 10 µl of each solution on the plate. 7. Leave your plate in the 30 C incubator overnight

5 Procedure Day 2: 8. Check the plates from yesterday and record your data in the table. 9. Participate in a class discussion about results. Take notes in the boxes below. Explain the results Analogies immunizations Winter jacket Prediction will it help to put the yeast in 37 C for 30 minute before transferring to the 51 C bath? 10. Label 2 test tubes with your group name and the following temperatures: 30 C and 37 C. 11. Put 300 µl of the culture provided by the teacher into each tube. Place each tube in the appropriate incubator or water bath for 30 minutes. 12. After 30 minutes, move both of your tubes into the 51 C water bath for 5 minutes. 13. After 5 minutes, remove the tubes from the 51 C bath and transfer them to the 30ºC bath until you re ready to dilute and plate. 14. You should dilute each sample by combining 100 µl of each sample with 900 µl of sterile YPD in a new labeled test tube. Mix the sample using a vortex or swirling. 15. Label two new YPD plates with your group name and either 30 C or 37 C. Inoculate a Petri dish of sterile YPD nutrient agar with each yeast culture. The yeast cells are suspended in a liquid nutrient broth. Using a disposable pipette, place 3-4 drops of the yeast laden broth on the agar of the Petri dish. 16. Use the sterile cotton swab to evenly (and gently!) disperse the broth across the entire surface of the agar. The liquid layer will not be thick, but make sure you have coated the entire plate. When done, place your cotton swab in the 10% bleach solution, cotton down. This will kill the yeast. 17. Place dishes in the 30 C incubator overnight. Procedure Day 3: 18. Observe results from yesterday s experiment. Count the number of colonies on each plate. 19. Participate in a class discussion about results. Take notes in the table below:

6 What are chaperone proteins? What are heat shock proteins? Data: Day 1 Sample Observations 30 C 37 C 51 C Day 2 Sample # Colonies Observations 30 C à 51 C 37 C à 51 C Analysis questions: 1. Did you see a difference in the ability of the yeast to survive in different temperatures (day 1)? 2. In your experiment from day 2, what was the independent variable? The dependent variable? The controlled variables? 3. Did you see a difference in the ability of the yeast to survive in 51 C based on whether or not they were first exposed to 37 C (day 2)? 4. Why was it important to sterilize all materials before running the experiment? 5. Based on what you learned from doing this lab, explain what you hypothesize causes the cell to produce heat shock proteins and explain what these proteins do under normal conditions and stressful conditions.