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23 Nov 2019

Recombinant expression of many mammalian proteins requiresproduction of the protein at below physiological temperatures (i.e.below 37^o C). If the protein is expressed at37^o C, all you produce is a large mass of aggregatedprotein called an inclusion body. If you express the same proteinat 20^o C, you produce a significant amount of soluble,functional protein.
Provide a thermodynamically based reason for why this would betrue.

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Creat Testable Question?

Not all questions are testable!!

A testable question asks. . .

"What is the effect of the Independent Variable on the Dependent Variable?"

Creat a Hypothesis?

A hypothesis is a prediction of the outcome of the lab. Your prediction is based on your understanding of the scientific concept.

It answers your testable question. Use the following pattern:

IF the (IV- how it is changing), then the DV- how you think it will change), BECAUSE (why do you think this is happening.

Example- If the IV increases, then the DV will decrease because....

6.Creat a Procedures ?

Procedures are a list of materials used in the experiment and the steps in performing the experimental part. Procedures are the recipe you follow in order to perform the experiment. Write these in order, in paragraph form, and using your own words. Do not simply copy and paste from the student lab guide.

7. Creat Data Table ?

Data is recorded during the procedures. You need to a data collection table for your experiment. Below is an example Data collection table. you need your own collection table using labels with the names of your variables and the correct units you are measuring.

{Insert name of Independent Variable} (insert units)

{Insert name of Dependent Variable} (insert units)

Average { Insert name of Dependent Variable }(insert units)

Trial 1 *

Trial 2 *

Trial 3*

Control

{Insert Treatment 1 Name}

{Insert Treatment 2 Name}

{Insert Treatment 3 Name}

Project Guide

Introduction from Vernier

Almost all chemical reactions that occur in living organisms are catalyzed by enzymes. Enzymes are globular proteins, responsible for most of the chemical activities of living organisms. They act as catalysts, substances that reduce activation energy, or the energy it takes to get a reaction started. Enzymes are not destroyed or altered during the process. They are extremely efficient and may be used over and over again. One enzyme may catalyze thousands of reactions every second. Many factors in a cell's environment, like temperature, pH, concentration and salinity, affect the action of an enzyme. Changes in these conditions may change the shape of the enzyme making it useless, a process called denaturation. In this investigation, you will conduct an experiment to determine the effect of a variable on an enzyme catalyzed reaction.

In every cell of the body, hydrogen peroxide, H₂O₂, is produced as a waste product. However, hydrogen peroxide kills living cells. Although H₂O₂ is toxic to most living organisms, many organisms are capable of enzymatically destroying the H₂O₂ before it can do much damage. H₂O₂ can be converted to oxygen and water, as follows.

2H₂O₂ â 2H₂O + O₂

Because your liver is responsible for detoxifying your blood, your liver cells contain a lot of catalase to break down H₂O₂ into harmless H₂O and O₂. Yeast cells also contain catalase, so we will use them as an easy and inexpensive source of catalase in this lab.

When the H₂O₂ encounters the yeast catalase, it bubbles, as the gaseous O₂ escapes. We will measure the amount of O₂ being produced using a gas pressure sensor. The more O₂ that is produced, the greater the pressure in the tube will become. An increase in pressure tells us that the catalase is effective at breaking down the H₂O₂.

The results of your experiment will be shared in an experiment report due week 15. This lab gives you hands on practice with the Vernier sensors. Use of the sensors will be required in other courses for the Natural Sciences Degree Program. The hands on procedure is detailed step by step below.

After reading through the direction make sure to watch the video showing the procedure in action.

Safety Precautions

Make sure to wear safety goggles. Keep a close eye on your pressures and make sure your gas sensor does not get wet (this will ruin the sensor)

Lab Materials

1 Vernier LabQuest

1 Vernier Gas pressure sensor w/ tubing set

1 Vernier Logger Pro (installed on computer)

1 Lab goggle (from HOL kit)

10 mL graduated cylinder (from HOL kit)

200 mL beaker (from HOL kit)

Tap water

4 cups (any kind)

1 Bottle FRESH 3% H₂O₂ (hydrogen peroxide)

3 18 x 150mm test tubes)

1 Test tube rack (create using cardboard, legos etc.)

1 packet of yeast

3 pipets (from HOL kit)

1 Thermometer (any kitchen or medical thermometer will work)

IMPORTANT NOTES:

You must use fresh hydrogen peroxide and yeast for this lab to yield decent results. Yeast can be bought at any grocery store (look at the expiration date closely) and hydrogen peroxide can be bought at any pharmacy or grocery store. You should buy a couple of packs of yeast in case you make an error in preparing the yeast solution If peroxide is kept in the refrigerator, it must be brought to room temperature before the lab.

Set out a large container of water so the water equilibrates to room temperature overnight.

Make sure you are using metric units on your thermometer. You can always run conversions from Fahrenheit to Celsius.

You must run a test on the catalase (yeast) to make sure that the yeast suspension is at the right concentration/activity before testing it with your independent variable. You should prepare the solution when you are ready to test your independent variable. The solution will serve as your control in the experiment

To make yeast suspension as seen in the included video, add the following into a cup :

100 ml warm water (37-40C, but no hotter or the yeast will die or enzymes will denature).

1 packet of yeast (approx 7g)

1 packet of sugar (the little packets they give at restaurants) (or make a 2% sugar solution by adding 4 g sugar to 250 ml water, then add a packet of yeast to 100 ml of this solution. You can use your scale provided in the HOL kit to weigh the sugar)

Let the solution sit for 10 minutes before testing.

Follow lab procedure steps 1 & 2. Add 2 drops of the yeast suspension and measure following lab procedure steps 4-9. (Make sure to read the lab steps before adding the yeast suspension. It is time sensitive and must be measured quickly after being dropped in the test tube.)

The room temperature reaction with two drops of suspension should produce around 130 kPa (1.3 atm) in 40-60 seconds. Pressures above 130 kPA will cause the stoppers to pop off. Pressures above 210 kPA may damage the sensor so keep a close eye on your numbers!

If your numbers are too high, add more warm water, to dilute the suspension. Start by adding a small increment of water (10 mL) and repeat the test. Continue adding water until you reach 130kPa in 40-60 seconds.

If the solution isn't strong enough, add 1 more drop to the test tube (3 drops instead of 2 for example). Continue to increase the number of drops until you get results near 130 kPA after 1 minute. Record the number of drops needed and use that number of drops as the control for the experiment. *With the exception of enzyme concentration, that will be the number needed for each test tube. If you are doing your experiment on enzyme concentration and it for example it takes 4 drops instead of 2 you will need to double the number of drops for all of your concentrations.

Variables:

You can modify this lab to test the effect of enzyme concentration, temperature, sugar source, enzyme source or salinity. Choose 1 independent variable to test. Below are lists of variables and some ideas on how to test them:

Enzyme: With 3ml H₂O₂ and 3ml H₂O in each tube, add 1 drop of enzyme suspension. Repeat with different numbers of drops. Make sure none of the enzyme solution touches the sides of the test tube and mix before measuring. These must be measured quickly after the drops have been added.

Temperature: With 3ml H₂O₂ and 3ml H₂O in each tube, add 2 drops of enzyme suspension. Fill cups with water of different temperatures. Set each test tube in the water for 5 minutes before recording data.

Salinity: With 3ml H₂O₂ and 3ml H₂O in each tube, add 2 drops of enzyme suspension. Add drops of 1M NaCl. To make 1M NaCL, dissolve 58.44grams of table salt in 1 liter of water. Repeat with different numbers of drops. These will also need to be measure quickly after the enzyme and NaCL solution are added.

Sugar source: Make the yeast suspensions with various types of sugars (brown sugar, granulated, powdered, glucose, fructose, lactose, etc). Measure quickly after yeast suspension is added to test tubes.

If you have an idea for another variable not on this list that you would like to test, please send me a message for pre-approval.

Procedure

Place test tubes in a rack and label them 1, 2, 3 etc. You can improvise a rack by cutting holes in a cardboard box. You just need something that will keep the test tubes vertical and easy to distinguish.

Add 3 mL of 3.0% H2O₂ and 3 mL of tap water to each test tube (Use a plastic pipet/dropper to easily dispense the water and H2O₂âdon't mix up the pipets!).

Conduct your experiment according to which variable you have chosen to test. Make sure to follow the time guidance for each variable as directed on page 3. For example if you were testing temperature you would place your tubes in water of different temperatures (pictured below), for salinity you would add drops of saline solution etc. If you have questions about how to test your chosen variable please send me a message.

To collect data connect the plastic tubing to the valve on the gas pressure sensor.

Connect the sensor to CH1 on the LabQuest.

Connect your Lab Quest to your computer. (You can conduct this experiment just using the Lab Quest but it is nice to use Logger Pro because you can easily export the graphs and tables for use in your report. ) Turn on Logger Pro (cd included with Vernier equipment, very easy to install on your computer and generates great graphics for your report)

In Logger Pro click Open â Experiments â Biology w Vernier â(Pressure)Enzyme.cmbl

Once Logger Pro is open to the Pressure Enzyme lab you should have all the lab settings pre-set so that the sensor will collect for the correct duration of time.

Turn the LabQuest on.

For your control, add 2 drops (or more depending on calibration) of the enzyme solution and put the stopper on the tube and GENTLY swirl the tube to mix the contents.

Start data collection by pressing the green play icon on your computer screen. The test will run for 180 seconds.

Disconnect the stopper from the test tube. Rinse out the test tube and leave it to dry.

Processing the DATA

There are several options for processing the Data. I recommend running through the steps listed in the experiment procedure on the Lab Quest. You can also follow the steps below to analyze the slope. The slope, m, tells us the pressure generated every minute, so the units would be kPa per minute, or kPa/min. Kilopascals, or kPa, are just a unit of pressure; you may have heard of other units of pressure like pounds per square inch (PSI) in car tires or atmospheres (atm) or millimeters of mercury (mmHG) for weather pressure. For example, if m is 0.00376 our rate of gas production is 0.00376 kPa/min. The larger the slope, the more oxygen is generated and the more active the enzyme is at those conditions.

Choose Curve Fit from the Analyze menu. Check mark Pressure.

Choose Linear Fit to get an equation in the form y=mx+b

Record the slope of the line, m, in your data table. Select OK.

Store the data from the first run.

Repeat steps 2-16 for each of your treatments.

Tap Run 3. Select All Runs. All three runs will now be on the same graph.

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