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Carbonic anhydrase is an enzyme that will convert CO2 and water into HCO3.

CO2 + H20 > H+ + HCO3

Important: Assume the Km of one variant of carbonic anhydrase is 1 µM. First, imagine an experiment employing 10 test tubes. The test tubes are all filled with 100 ml water. Imagine that the different test tubes have different concentrations of CO2, ranging from 0.1 µM to 1 mM CO2. Initially, there is NO enzyme in any of the test tubes.

(a) Draw a graph showing what you might expect to see for the amount of HCO3 in each of the test tubes 1 minute after the addition of CO2. Be sure to label the axes of the graph. Designate this curve as “A” (note: the exact values on the Y axes here are guestimates – you get to make them up).

(b) Now, imagine that the test tubes are filled with water and a significant amount (say, 10 mM) of the enzyme carbonic anhydrase in each test tube. Draw on the same graph as before what you might expect to see for the amount of HCO3 in each of the test tubes 1 minute after the addition of CO2. Label this curve “B”. (note: again, the exact values on the Y axes here are guestimates – you get to make them up).

(c) Now, on the same plot, draw a graph showing the curve that results when you subtract the data obtained in the first condition (without enzyme) from the data obtained in the experiments obtained when the enzyme is present. Label this curve “C”.

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Trinidad Tremblay
Trinidad TremblayLv2
29 Sep 2019

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Related questions

1. Carbonic anhydrase is an enzyme that willconvert CO2 and water into HCO3.

CO2 + H20 > H+ + HCO3

Assume the Km of one variant of carbonicanhydrase is 1 mM.

First, imagine an experiment employing 10 testtubes. The test tubes are all filled with 100 ml water. Imaginethat the different test tubes have different concentrations of CO2,ranging from 0.01 mM to 100 mM CO2. Initially, there isNO enzyme in any of the test tubes.

(a) Draw a graph showing what you might expectto see for the amount of HCO3 in each of the test tubes 1 minuteafter the addition of CO2. Be sure to label the axes of the graph.Designate this curve as %u201CA%u201D (note: the exact values onthe Y axes here are guestimates %u2013 you get to make them up). (4points)

(b) Now, imagine that the test tubes arefilled with water and a significant amount (say, 1 mM) of theenzyme carbonic anhydrase in each test tube. Draw on the same graphas before what you might expect to see for the amount of HCO3 ineach of the test tubes 1 minute after the addition of CO2. Labelthis curve %u201CB%u201D. (note: again, the exact values on the Yaxes here are guestimates %u2013 you get to make them up). (2points)

(c) Now, on the same plot, draw a graphshowing the curve that results when you subtract the data obtainedin the first condition (without enzyme) from the data obtained inthe experiments obtained when the enzyme is present. Label this curve %u201CC%u201D. (2 points)

4. Using the data from 2C above (that is, subtracting thebackground production of CO2 due to simple mass action from thatresulting from the combination of enzyme action and mass action),draw a graph showing the alteration in the rate of carbonicanhydrase as the CO2 level is varied as a percentage of the maximal rateof HCO3 production %u2013 that is, 100% is as fast as the enzymecan function. Be sure to label the axes of the graphs. That is,again draw a graph showing what you might expect to see for theamount of HCO3 produced by the action of the enzyme in each of thetest tubes 1 minute after the addition of CO2. (4 points)

There are many different isoforms of thisenzyme (see for instance

http://en.wikipedia.org/wiki/Carbonic_anhydrase

Assume that one variant has a Km of 10 mM.Draw on the same graph as above a plot showing the alteration inthe rate of carbonic anhydrase as the CO2 level is varied, assumingthe concentration of the enzyme (1 mM) in the test tube is the sameas for the previous enzyme. (2 points)

Experiment 1 Fermentation by Yeast Experiment Inventory Labware (4) 250 mL Beakers (1) 100 mL Graduated Cylinder (1) Test Tube Rack (5) Fermentation Tubes = (10) Test Tubes (5 plastic and 5 glass; see Figure 4) (1) Measuring Spoon (4) Pipettes (1) Ruler Note: You must provide the materials listed in *red. EXPERIMENT 1: FERMENTATION BY YEAST Yeast cells produce ethanol, C2 H6 O, and carbon dioxide, CO2 , during alcoholic fermentation. In this experiment, you will measure the production of CO2 to determine the rate of fermentation in the presence of different carbohydrates with fermentation tubes. Note: Regular table sugar is sucrose, a disaccharide, which is made up of glucose and fructose. Glucose is a monosaccharide. Figure 4: Fermentation tubes. Note how the smaller, plastic test tube is inverted into the larger glass tube. You will create five fermentation tubes in this experiment. PROCEDURE 1. In this experiment, you will mix yeast with sugar, Equal®, and Splenda®. Before you begin, develop a hypothesis predicting what will happen when the sugar/sweeteners are mixed with yeast. Will fermentation occur? Why or why not? Record your hypothesis in the post-lab questions. 2. Use the permanent marker to label three 250 mL beakers as Equal®, Splenda®, and Sugar. 3. Empty the Equal®, Splenda®, and Sugar packets into the corresponding beakers. 4. Fill the Equal® and Splenda® beakers to the 100 mL mark with warm tap water. 5. Fill the Sugar beaker to the 200 mL mark with warm tap water. 6. Mix each beaker thoroughly by pipetting the solution up and down several times. Use a new pipette to mix each solution. Each beaker now contains a 1% solution. Set these aside for later use. 7. Completely fill one of the smaller plastic tubes with tap water and invert the larger glass tube over it. Push the small tube up into the larger tube until the top connects with the bottom of the inverted tube. Invert the fermentation tube (Figure 4) so that the larger tube is upright (there should be a small bubble at the top of the internal tube). Note: Repeat Step 7 several times as practice. Strive for the smallest bubble possible. When you feel comfortable with this technique, empty the test tube(s) and proceed to Step 8. CAUTION: Do not try to force the plastic test tube into the glass test tube. This might cause your glass test tube to break, causing you injury. If your plastic test tubes do not fit easily, please call eScience Labs for replacement glass tubes. If you are able to set up at least two fermentation tubes, continue with the experiment, but know that you will have to perform steps 12-15 in multiple steps. 8. Use the permanent marker to label the fourth 250 mL beaker as Yeast. 9. Fill this beaker with 175 mL of warm tap water. It should be between 30 and 40o C (warm to the touch). 10.Open the yeast package, and use the measuring spoon to measure and pour 1 tsp. yeast into the beaker. Pipette the solution up and down until all of the yeast is mixed homogenously into the solution. Note: Make sure the yeast solution remains homogenous before each test tube is filled in the proceeding steps. The yeast density is fairly high, and the yeast may settle to the bottom of the beaker if it rests for an extended period of time. 11. Use the permanent marker to label the big glass and small plastic test tubes as 1, 2, 3, 4, and 5. 12.Use the 100 mL graduated cylinder to measure and pour 15 mL of the following solutions into the corresponding small plastic test tubes: Tube 1: 1% Glucose Solution Tube 2: 1% Sucrose Solution Tube 3: 1% Equal® Solution Tube 4: 1% Splenda® Solution Tube 5: 1% Sugar Solution Note: Thoroughly rinse the graduated cylinder between each measurement. 13.Fill the remaining volume in each small tube to the top with the yeast solution. 14.Slide the corresponding larger tube over the small tube and invert it as practiced in Step 7. This will mix the yeast and sugar/sweetener solutions. 15.Place the fermentation tubes in the test tube rack, and use a ruler to measure (in millimeters) the initial air space in the rounded bottom of the internal tube. Record these values in the Table 1. 16.Allow the test tubes to sit in a warm place (approximately 30 °C) for two hours. Placement suggestions include: a sunny window sill, atop (not in!) a warm oven heated to approximately 85 °C (185 °F on an oven setting), or under a very bright (warm) light. 17.At the end of the fermentation period, use your ruler to measure (in millimeters) the final gas height (total air space) in each tube. Record this data in Table 1. 18.Calculate the difference between the initial and final gas height in each tube. Record this data in Table 1.

EXPERIMENT 1: FERMENTATION BY YEAST

Result Tables

Table 1: Yeast Fermentation Data

Tube

Initial Gas Height (mm)

Final Gas Height (mm)

Net Change (mm)

1

2

3

4

5

Post-Lab Questions

Include your hypothesis from Step 1 here. Be sure to include at least one piece of scientific reasoning in your hypothesis to support your predictions.

Did you notice a difference in the rate of respiration between the various sugars? Did the artificial sugar provide a good starting material for fermentation?

Was anaerobic fermentation occurring? How do you know (use scientific reasoning)?

If you observed respiration, identify the gas that was produced. Suggest two methods you could use for positively identifying this gas.

Hypothesize why some of the sugar or sweetener solutions were not metabolized, while others were. Research the chemical formula of Equal® and Splenda® and explain how it would affect yeast respiration.

How do the results of this experiment relate to the role yeast plays in baking?

What would you expect to see if the yeast cell metabolism slowed down? How could this be done?

Indicate sources of error and suggest improvement (for example, what types of controls could be added?).

Please answer questions at the end.

In this lab, you will add different volumes of “cell-free supernatant” (CFS) to fresh cultures of V. harveyi and observe bioluminescence. The CFS is prepared by culturing V. harveyi in liquid medium overnight, then separating the cells from the supernatant by centrifugation.

MATERIALS • overnight culture of V. harveyi in AB medium • fresh AB medium • 1 microcentrifuge tube • 3 glass culture tubes

PROCEDURE

1. This experiment will be performed with your lab group. Label the microcentrifuge tube and 3 glass cultures tubes with your group number. Also, label the 3 glass culture tubes as follows: “Media only,” “Cells+fresh media,” and “Cells+CFS.”

2. Using aseptic technique, pipet different volumes of fresh AB medium into the glass tubes as follows: 1 mL in the “Media only” tube, 800 ul in the “Cells+fresh media” tube, and 500 ul in the “Cells+CFS” tube.

3. Pipet 500 ul of the overnight culture into a microcentrifuge tube and centrifuge at maximum speed (>13,000 x g) for 5 min to pellet the cells.

4. After centrifugation is complete, transfer 300 ul of the cell free supernatant (CFS) from the microfuge tube to the glass tube labeled “Cells+CFS”. Be careful to avoid the cell pellet.

5. Remove the remaining supernatant from the microfuge tube using a micropipette and discard as culture waste.

6. Add 500 ul of fresh AB medium to the cell pellet in the microfuge tube and pipet up and down to resuspend until no clumps are visible.

7. Add 200 ul of the cell suspension to the “Cells+fresh media” tube and 200 ul of the cell suspension to the “Cells+CFS” tube. Note the time.

8. Place the 2 glass tubes containing cells in the 30 degree incubator/shaker.

9. After 1 hour and 2 hours, observe your tubes next to the “Media only” tube in a dark room. You will need to let your eyes adjust for at least 30 seconds (count to 30 slowly in the dark) before you can evaluate whether the cultures are luminescent or not. Also, make sure you try swirling the tubes in the darkroom and observe the tubes for a minute after you have stopped swirling. After the observation at the 1 hour time point, put your tubes back in the incubator shaker until the 2 hour time point.

10. Note your observations. At the end of the lab, place your tubes in the tube waste.

Questions

1. What were the expected results? Explain the rationale of the experiment. If you didn’t obtain the expected results, hypothesize why not and describe how the experiment could be improved.

2. What are differences between luminescence and fluorescence?

3. Why do V. harveyi cultures glow more brightly when swirled vigorously? Be specific.

4. Imagine you have a mutant V. harveyi strain that can’t synthesize autoinducer and another mutant V. harveyi strain that makes a defective luciferase. If you streaked both strains near (but not touching) each other on the same plate, would you expect to see light produced? If so, by which strain, where, and why?

5. You have two identical tubes of wild-type V. harveyi (with the same cell concentration in each tube) in fresh media. To one tube, you add CFS from a light-producing V. fischeri culture, and to the other tube you add an equal volume of fresh media. If both tubes of V. harveyi have the same light production, what can you conclude? Explain.

6. Many biomedical research labs study quorum sensing in V. fischeri or V. harveyi. How is studying light production in marine bacteria relevant to human health? List at least 3 different examples.

7. Why do you think it is important for bacteria to be able to control the expression of some genes based on the number of individuals present?

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