Enzymes: Planning Your Project

Laboratory Inquiry 4:

Enzymes: Planning Your Project

IMPORTANT: Youmustbring a laptop or other electronic device for taking notes and keeping a copy of your team’s Project Planning Worksheet and other team materials completed in this lab session.

Your team has spent one session hammering out ideas for an experiment to study the nature of catalase. In today’s section, you will formalize and finalize your project. By the end of today’s lab session, your team should

a. have a good, working overall hypothesis

b. make a prediction with respect to your overall hypothesis

c. have null and alternative experimental/statisticfal hypotheses

d. have a complete, step-by-step protocol including instructions for preparing reagents

in specific quantities and concentrations, as necessary.

I. Visualizing your ideas

Your team should use theProject Planning Worksheet you prepared last week to create a PowerPoint presentation consisting of exactly THREE SLIDES. For each presentation:

Slide #1. Clearly state your research question

Slide #2. Create a visual representation of your research.

Slide #3. State what you think your results will be.

This exercise will help you focus on the most important aspects of your research: the IDEA, the overall idea of the protocol, and what you expect to see.

If you’re not sure about Slide #2, see the two examples in Figures 1 and 2 below.

Figure 1. Francesco Redi tested the idea of spontaneous generation (that life could spring from non-living matter, and in this specific case that maggots could spring, fully formed, from rotting meat) with the experimental set up shown here. In Treatment 1, he placed meat in uncovered jars. In Treatment 2, he placed meat sealed jars. In Treatment 3 he placed meat in jars covered with a screen that would allow air into the jar, but not much else. Each Treatment yielded different results.

Your lab instructor will give the teams about 30 minutes to create these slides, and then deliver them to the front desk via flash drive (or email, or whatever method your instructor chooses). When all teams are ready, each team will give a short presentation of their slides, and be critiqued by both the lab instructor and your colleagues (fellow lab students).

II. Refining Your Experiment

Once your proposal has been critiqued by your colleagues, you should be ready to go back to the drawing board and refine your ideas and your protocol. Use the Project Planning Worksheet(PPW) (linked to the syllabus) again, and complete a new one to reflect your revision.

A. Observation, Hypothesis, Prediction

When your team has revised its observation, overall hypothesis, and prediction to all members’ satisfaction, bring your PPW to your lab instructor for final approval. If your instructor approves this first step, you are ready to move on and design your methods.

B. Materials

1. How many runs, each, of your treatment and control groups will you run?

Treatment:

Control:

2. You will be supplied with the following standard reagents:

Stock yeast suspension (70g yeast/L pH 7 sodium phosphate buffer)
Stock hydrogen peroxide (33mL of 9.1% H2O2/L pH 7 sodium phosphate buffer)
Stock sodium phosphate buffer (0.05M, pH 7)
Deionized (DI) water

Remember that each control reaction is run with 10mL of standard yeast suspension and 20mL of H2O2 in pH 7 buffer. With this information, calculate the amount of each of the stock solutions of yeast, peroxide, buffer, and water your team will need.

Name / Description (e.g., molarity, pH, etc.) / Quantity/volume
Yeast suspension / 70g/L in pH 7 sodium phosphate buffer
Hydrogen peroxide / 9.1% H2O2/L in pH 7 sodium phosphate buffer
NAPO4 buffer / pH 7.0, 0.05M
water / deionized

3. You will have from among the following special reagents to choose for your experiment.

Recall that your literature search—and those of many of your colleagues—revealed that much research done on catalase involves chemicals that pose a safety hazard. While we feel it is important for you to have read these papers and thus learned more about catalase (and other, incidental things that could prove interesting and helpful), we also feel it is important that you end this semester alive and well. To this end, we will provide a variety of stock chemicals from which your team may choose to design its experiment. They are as follows:

a. ascorbate solution, 0.1M (100mL brown or foil-wrapped bottle)

b. succinate solution (0.04M) (100mL covered glass vessel)

c. copper sulfate solution (0.1M) (100mL covered glass vessel)

d. acetyl salicylate solution (10mM) (100mL in covered glass vessel)

e. sodium chloride solution (1M) (100mL in covered glass vessel)

f. 95% ethanol in covered glass vessel

g. 100% isopropanol in covered glass vessel

(NOTE: a solid chemical’s name is often different from that chemical’s name in solution. For example, ascorbic acid is a solid. In its dissolved, ionized form, it is known as ascorbate. The same is true for succinic acid/succinate. So don’t let the names above confuse you.)

Remember: just because we provide a chemical for you does not mean that we’re going to tell you how it works or why it would be interesting to test its effects on catalase. It is up to your team to explain its rationale. But choose only from among those in the list above.

In addition to the stock solutions from #2, what reagents will you need, and how much?

List them here.

Name / Description (e.g., molarity, pH, etc.) / Quantity/volume

4. If your protocol requires that you prepare reagents not supplied as stock solutions or

solids, calculate how you will prepare each of your reagents. Use these formulas to

calculate concentrations and dilutions.

a. To calculate the mass (g) of a solid chemicalneeded to make a solution of a desired volume

and concentration of Chemical X, use this equation:

(desired concentration in moles/L) x (desired volume in L) x (molecular weight of Chemical X in g/mole)

= g of Chemical X needed

b. If you have a stock solution of known molarity and you want your final yeast suspension to be a

different molarity, use this equation:

(concentration of stock solution in moles/L)(volume of stock solution in L)

= (desired final concentration of NaCl in your yeast suspension)(desired final volume)

Remember: If the amount of yeast suspension and the value of x do not add up to your desired final volume, you should add deionized water to your suspension to achieve the desired final volume so that treatment and control are the same.

Create a step-by-step “recipe” for each reagent you plan to prepare. Anyone should be able to follow your directions to replicate your work. Use this template, or create one of your own. But each team member should have a copy to take home at the end of lab.

Reagent #1:

10.

Reagent #2:

If you are examining the effect on catalase of an environmental variable (e.g., temperature, pH, concentrations, you will not need chemical reagents beyond the stock solutions in #2. However, you still must create a protocol.

5. Using this template, or one of your own design, create a detailed, step-by-step protocol for your particular needs.

We have posted this lab chapter as an MS Word Document so you can easily modify the Sample Experimental Protocol you used to test the effect of NaCl on catalase. The Sample Experiment protocol appears below, exactly as you followed it. Read it carefully, as a team, and modify it precisely to fit your own experimental needs.

III. Designing your Protocol

Below, you will find the Sample Experimental Protocol you used in Session 2. Modify it here (it’s in MS Word, not a .pdf), and submit the completed protocol to your instructor when you are finished.

Wherever you modify the protocol, change the color of the font to BLUE. This will allow your instructor to easily note the methods you are modifying for your own experiment.

Modify this Protocol:

1. Check workstation and equipment. Clean any dirty items with deionized water and drain on a paper towel. (Check the list of supplies, above, that should be at your station. These items must be clean and back at your station when you check out for the day or your grade will be docked. When you are ready to leave, ask your Undergraduate Teaching Assistant (UGTA) to check your station and approve it before you go.)

2. Using labeling tape and a marker, re-label syringes and other supplies, as necessary. Remember: If an item is clean, it doesn’t need a label until you have contaminated it with a reagent. If any item (syringe, graduated cylinder, beaker, etc.) touches a solution, it MUST be labeled with the type of solution (concentration and chemical) and your team name (in case items may get mixed up between neighboring groups). Clean and re-label items throughout the lab as necessary.

3. Open the LoggerLite program on the laptop your team will use for data collection. (The download link is on page 3 of this chapter.)

4. Keeping the O2 sensor probe UPRIGHT in its box, connect it to the laptop using the adaptor cable. Remember to keep the probe dry and upright in its labeled beaker at all times when not in use.

5. To ensure that the sensor is working correctly, click “Collect” on the toolbar. The %O2 should be recorded once per second in the table to the left and on the graph. Try breathing into the dry plastic respiration chamber and putting the sensor in the top. The oxygen readings should go down. Now click “Stop.” You can click “store” on the toolbar to save one data run and “save” to save an entire batch. To open a fresh data table and graph, click “New.”

6. Before beginning the experiment, delegate duties to everyone in the team. Every person must know how to do every task, but it will be easiest to assign duties such as manning the computer, cleaning glassware, recording times, and so on amongst the team members.

A. Preparing your Treatment Samples

Since the treatment groups will need time for the salt to induce a response in the yeast, those solutions should be set up first.

1. Label three beakers Treatment 1, 2, and 3

2. Find the stock yeast suspension on a tray at the center of your lab table. Mix the contents to

ensure that yeast are evenly distributed and then decant 70mL into a labeled beaker. Stir the suspension with a stirring rod often to keep yeast from settling.

3. Add 10 mL of yeast suspension to each Treatment beaker, using the labeled syringe.

4. Add 4.5 mL of 1M NaCl and 0.5 mL of water to the first beaker and record the time on the

beaker label. Set a timer or alarm on your phone for 30 minutes. (When the alarm goes off, it’s time to use Treatment 1 for an experimental run.)

5. Wait five minutes. Then repeat steps 2, 3, and 4 to prepare Treatment 2.

6. Wait an additional five minutes. Then repeat steps 2 and 3 again to prepare Treatment 3.

(Waiting five minutes between each treatment preparation will allow you to stagger the times when the samples are ready to be used, and still have the same NaCl exposure time for all treatment samples.)

B. Preparing and Using your Control Samples

After the treatment groups are set up, the control groups can be prepared. Since they do not need to be incubated, you may place each control sample directly into the Vernier plastic respiration chamber and run the experiment immediately, without placing your samples in separate beakers.

1. Mix your remaining yeast suspension to ensure that yeast are evenly distributed.

2. Add 10 mL of yeast suspension to the plastic Vernier respiration chamber.

3. Add 5 mL of deionized water to ensure equal volumes between the treatment and control

groups.

4. Find the stock hydrogen peroxide solution on the tray at the center of your lab table. Decant

about 135 mL into a labeled beaker. Even at this low concentration, hydrogen peroxide is slightly caustic, and can bleach clothing. Don’t splash it!

5. Back at your station, draw 20mL of your H2O2 solution into a labeled syringe.

6. Prepare everything for an experimental reading, making sure your LoggerLite software is

ready, that everyone is in position and ready to do his/her job.

7. When everyone is ready to start the experiment, quickly and carefully squirt 20 mL of H2O2

solution into the plastic Vernier chamber already containing your yeast suspension.

8. Quickly and carefully insert the O2 probe to seal the chamber.

9. Gently swirl the container (hold it by the neck, not the body) to stir. Do not rub the bottom of

the chamber on the tabletop, to avoid friction. Swirl just enough to keep the mixture moving, but don’t wet the probe!

10. The reaction begins very shortly after the introduction of hydrogen peroxide, so click

“Collect” in the LoggerLite program once you have begun swirling your suspension. Be very careful to keep the probe dry or your readings will become erratic. If this happens, use one of the ringstand/fan assemblies set up along the back table to dry your probe. Do not clamp the probe tightly, to avoid damaging it.

11. When the %O2 readings begin to plateau, click “Stop.” This will happen automatically after 300

seconds.

12. Click “Store” to save the readings from that sample.

13. Gently remove the O2 sensor and place it upright in its dry, labeled beaker.

14. Discard the yeast suspension in the front or rear sink and wash the chamber well with tap

water until it it completely rinsed and clear. Give it a final, all-over rinse with deionized water three times to ensure that no residue is left behind. DO NOT POUR YEAST SUSPENSION INTO THE LAB BENCH SINKS! THESE HAVE BEEN DE-COMMISSIONED DUE TO YEARS OF ABUSE BY NEGLIGENT STUDENTS, AND YEAST CANNOT BE RINSED DOWN. THEY WILL SIMPLY SIT AND FERMENT IN THE PLUMBING, CAUSING HORRIBLE STENCH, AND GREAT WAILING AND GNASHING OF TEETH.

15. Repeat Steps 10-14 twice more for the remaining control groups. Work quickly and carefully in

order to be ready for the treatment groups when their 30 minute incubation time is up (i.e., when your first alarm goes off).

16. Follow Steps 1-13 for each of your treatment groups, substituting your NaCl samples for the

stock yeast suspension. All other steps in the protocol should be the same as for the control groups. (Note: Do not add 0.5mL of DI water to treatment samples; only controls.)

C. Data Analysis

What statistical test will you use to analyze your results?

D. Before you Leave…

At the end of lab, each team member should receive a copy of all the calculations you have done above, as well as the modified protocol.

Your lab instructor should be given an electronic copy of this lab manual chapter with your modifications, and must give it final approval before any of your team members leave the lab.

Leave without instructor approval, and each team member will lose 5 points.