Lab 8.pdf

LABORATORY EXERCISE 8 Carbon Metabolism by Yeast Learning Objectives * Determine if the structure of a carbon source impacts cellular metabolism * Consider why yeast metabolize some carbon sources more efficiently than others * Understand the strengths and weaknesses of different measurement techni...

LABORATORY EXERCISE 8 Carbon Metabolism by Yeast Learning Objectives * Determine if the structure of a carbon source impacts cellular metabolism * Consider why yeast metabolize some carbon sources more efficiently than others * Understand the strengths and weaknesses of different measurement techniques This exercise will explore the catabolism of different carbon sources by yeast. Yeast are unicellular eukaryotic organisms that are able to metabolize some carbon sources (including sugars), but not others. Through catabolic pathways, cells break down sugars and other macromolecules into smaller components. They do this because 1) those smaller components can be reconfigured into larger molecules through anabolic pathways and 2) they extract energy stored in the bonds of the sugars and macromolecules. To accomplish this, the macromolecules must first be transported into the cells, and the cell must possess the proper enzymes capable of breaking/altering these molecules’ chemical bonds in a useful way. Yeast can metabolize carbon sources using two major catabolic pathways called aerobic respiration and fermentation. Aerobic respiration requires oxygen (O2) and carbons metabolized in this pathway are fully oxidized to form carbon dioxide (CO2) as a byproduct. The metabolic pathway of aerobic respiration preferentially uses glucose as a carbon source. After transport into the cell, glucose is broken down via glycolysis, which is then followed by the citric acid cycle and oxidative phosphorylation. The net result of this process is the production of cellular energy in the form of ATP (Fig. 7.1). The final steps of oxidative phosphorylation require O2 to serve as the final electron acceptor. This entire catabolic process explains why humans and other aerobic organisms breathe in O2 and exhale CO2 at a cellular level. Fig 7.1 © Pearson Education, Inc. Freeman 2011 Fig 7.2 © Pearson Education, Inc. Freeman 2011 In contrast to aerobic respiration, fermentation does not require O2. In yeast, fermentation can occur utilizing specific carbon sources that are typically referred to as fermentable carbons. When yeast metabolize fermentable carbons, they produce ethanol and CO2 (Fig. 7.2). Humans will produce lactic acid when undergoing fermentation. If yeast are grown in the presence of a non-fermentable carbon such as glycerol, they must undergo aerobic respiration. In this exercise, you will determine how effectively Baker’s yeast metabolize different carbon sources. Whether yeast are using aerobic respiration or fermentation, they will produce CO2. Using a CO2 gas sensor, you will measure CO2 production by yeast incubated with four different carbon sources. After collecting these measurements, you will examine yeast cells under the microscope to correlate carbon metabolism to the rate of cellular division. The four carbon sources you will use in this experiment are: Glucose-monosaccharide (blood sugar) Glycerol- non-fermentable carbon source (not a sugar) Sucrose-disaccharide of glucose and fructose (table sugar) Lactose-disaccharide of glucose and galactose (milk sugar) What do you notice about all of these carbon sources? How are they similar? How are they distinct? Compare the disaccharide carbon sources. What is major differences do you notice when comparing these? Which carbon source do you predict yeast will metabolize the best? Which carbon source will get metabolized the worst? Will all carbon sources get metabolized equally well? Write your statement below and provide justification for why you think this is true. Instructor demonstration Your instructor will prepare a demonstration in which yeast are exposed to each carbon source in a large Erlenmeyer flask. Prepare each flask by mixing no less than 50mL of yeast solution + 50mL of sugar solution. These volumes should produce a robust response. Once the flasks are prepared, affix a balloon to the top of the flask and incubate each in the 35° C water bath. Students should observe the balloons after they have incubated for ~1 to 1.5 hours in the water bath. Have students take a picture of the balloon results to compare against their group data and class results. Procedure 1. Label four beakers as follows: glucose, sucrose, glycerol, and lactose. Obtain approximately 5 ml of each solution and take them to your workbench. Each carbon source solution is prepared to a 10% final concentration. 2. Label six test tubes as follows: G, S, Gy, L, L2, and W. 3. Pipette the following in each test tube: a. 2 ml of glucose solution in G b. 2 ml of sucrose solution in S c. 2 ml of glycerol solution in Gy d. 2 ml of lactose solution in L e. 2 ml of lactose solution + ¼ crushed Lactaid™ tablet in L2 f. 2 ml of water in W What is the purpose of the W tube? Why is this tube necessary for the experiment? How do you predict yeast will behave when exposed to water? 4. Obtain ~15 ml of yeast suspension that is warmed up in the water bath. Gently swirl the suspension prior to obtaining since yeast will settle to the bottom. 5. Once again, swirl the suspension while adding 2 ml of yeast into each test tube. Once the yeast has been added, gently mix the test tubes. 6. Place the test tube rack containing your yeast and sugar mixture into the warm water bath. Incubate for 10 minutes. What is the purpose of letting the yeast incubate for 10 minutes? 7. Prepare your CO2 sensor and metabolic chamber while the yeast are incubating. First, set the CO2 sensor switch to the Low setting (0-10,000 ppm). The switch is located by the red CO2 label. Connect the sensor to LabQuest and choose “New” from the File menu. 8. When the 10 minute incubation is finished, observe each test tube in the rack. Do all test tubes appear the same? Describe the appearance of each test tube below. How do you predict the appearance of each test tube will reflect the measurements you are about to make using the CO2 sensor? 9. Once you have recorded your observations, select one test tube and pour the reaction mixture into your metabolic chamber. Keep the other test tubes in the water bath while you are measuring your first experiment. * Record the order in which you select your test tubes. This will become important when performing the data analysis step. 10. Place the probe from the CO2 gas sensor into the opening of the metabolic chamber (Fig. 7.3). Fig 7.3 CO2 sensor and metabolic chamber 11. Wait one minute for the readings to stabilize. 12. After one minute, record the CO2 ppm as shown on the LabQuest as your t=0 timepoint. Continue recording CO2 measurements every 20 seconds for a 4-minute time span in Table 7.1 below. The Table is large and wraps around the document! 13. Once you have finished collecting data from the first sample, remove the CO2 sensor from the metabolic chamber and thoroughly rinse the chamber clean. Make sure all yeast has been removed from the chamber and dry it with a paper towel. 14. Fan air over the CO2 probe using a notebook for 1 minute to reset it to zero. 15. Repeat steps 9-14 for each mixture you prepared. Data Processing 1. First, subtract your t=0 measurement from each subsequent ppm measurement for each sample. This is called data normalization. Why is it necessary to subtract the t=0 measurement from each sample? Why do you think this step is referred to as ‘data normalization’? 2. Plot your data for each sugar on an X-Y scatterplot. You should plot time (seconds) on the X-axis and the normalized CO2 ppm measurements on the Y-axis. Draw a best-fit line for each carbon source. Plot each different carbon source on the same graph so that you can directly compare them. Describe the best-fit lines for each carbon source. How do their slopes compare? What can you conclude based on the slope? Does this conclusion match your initial prediction? 3. Calculate the metabolic rate (ppm of CO2/s) between two points by calculating the slope. Try to identify the most linear point of your best-fit line for this calculation. The following equation can be used for the slope: (ppm2-ppm1)/(time2-time1). Record your data in Table 7.2. 4. Report your averages on the board and calculate the average metabolic rate with each value’s standard deviation. Record these values in Table 7.3. How much variation do you observe in the metabolic rates from class data? What do you think is the major contributor to the standard deviation? 5. Plot a bar graph of the metabolic rate with the rates on the y-axis and each carbon source on the x-axis. Rank each metabolic rate from best (1) to worst (6). How do the ranks compare between each lab group? Which carbon source was metabolized the best? Which carbon source was metabolized the worst? How do these observations compare to your predictions? Based on your measurements, which balloon from the instructor demonstration do you predict will be the most inflated? Which will not be inflated? Data Time (s) Glucose Sucrose Glycerol Lactose Lactose + Lactaid™ Water Time (s) Glucose Sucrose Glycerol Lactose Lactose + Lactaid™ Water 0 Table 7.1 CO2 ppm for each carbon source 20 40 60 80 100 normalized normalized normalized normalized normalized normalized 140 Table 7.1 CO2 ppm for each carbon source 160 180 200 220 240 normalized normalized normalized normalized normalized normalized Metabolic rate calculations Glucose: 120 Sucrose: Glycerol: Lactose: Lactose + Lactaid™: Water: Table 7.2- Lab group results Carbon Metabolic Rate source (ppm/s) Glucose Sucrose Glycerol Lactose Lactose + Lactaid™ Water Table 7.3- Class average metabolic rates Carbon source Metabolic Rate (ppm/s) Glucose Sucrose Glycerol Lactose Lactose + Lactaid™ Water Observe the Instructor demonstration At this point, the instructor demonstration should be complete. Describe what you see for each balloon and carbon source below. Questions Standard Deviation 1. Describe the relationship between yeast metabolism of different carbon sources with both CO2 production and cell division. 2. Why do you think yeast can metabolize some carbon sources better than others? 3. What does the Lactaid™ do to lactose? How would this help people that are lactoseintolerant? 4. Describe one new thing that you learned from today’s experiment. 5. Provide one follow-up question that you could address using a similar experimental approach. How would you set up this experiment to test your idea?