Lab 8: Pre-Lab

Your task in Lab 7 is to design and carryout an experiment regarding the rate of photosynthesis. You will complete a research proposal and analyze your data as part of this lab. To prepare for Lab 7, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding pre-lab quiz in Blackboard.

• Introduction/Review
• Do you know enough?
• What will we do in lab?
• LABridge

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Photosynthesis & The Light Reactions 

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Review & Connections: Energy is the currency of life: all living organisms require energy to survive and reproduce. Metabolism is the series of reactions and processes, catalyzed by enzymes, which together maintain life. These reactions fall into two types: catabolic or anabolic. These processes are the inverse of each other and in photosynthetic organisms occur in tandem as the anabolic reactions of photosynthesis create the products that are then broken down by the catabolic reactions of cellular respiration. 

Photosynthesis is a light-dependent process in which radiant energy is captured, and converted to chemical energy which is then utilized to create fuel for organisms. Chlorophyll A is the primary photoreceptive pigment and is adept at absorbing wavelengths in the blue and red spectrum, but wavelengths in the green and yellow (500-600 nm) are transmitted, thus, most plants appear green. You should know about the other secondary pigments and with what colors they are associated.
​There are two primary sets of reactions in photosynthesis.

1. The Light Reactions:  The mechanism behind this important process starts with photoreceptive pigments within the chloroplasts of plant cells. When these pigments absorb light energy, they become excited, and along with the energy from splitting water, the process generates adenosine triphosphate (ATP) and NADPH, chemical forms of energy. *We will be measuring this process! Specifically, we will measure how quickly NADP+ is reduced to NADPH.
2. The Calvin Cycle: The ATP and NADPH are then used to reduce CO2 and assemble glucose (C6H12O6), a common monosaccharide and major energy source for most living organisms. Often plants assemble glucose into the more complex polysaccharide of starch. Without the ability of photosynthesis to convert light into fuel, heterotrophs (all animals including humans) would cease to exist. 

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CONNECTION! ​​Photosynthesis chapter 10 in your BIOL 120 lecture. Please review your textbook as needed for this lab. The most important thing you need to know for Lab 6, are the reactants and products of aerobic cellular respiration. 

Please review chloroplasts, the light reactions and the Calvin cycle.

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Review of the light reactions  

Review of the calvin cycle          

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Chloroplast diagram. Click to enlarge.

2- Do you know enough about spectrophotometry?

Wavelengths of light are associated with various colors due to their frequency.  When light hits an object or solution (like a plant leaf in the figure below), wavelengths are either absorbed or reflected/transmitted. We see color based on the associated wavelengths that are transmitted or reflected. We do not see the colors associated with the wavelengths that are absorbed.

We see the color of the wavelengths that are transmitted or reflected. We do not see the colors of wavelengths that are absorbed.

For example, view the absorption spectrum for typical plant pigments (graph below). The graph shows that when light hits a plant, purple, blue, red, and orange wavelengths have high absorbance values, meaning they are mostly absorbed by the plant and we don not see these colors. Green and yellow wavelengths have low absorbance values- meaning they are reflected... this is what gives plants their green color! 

Absorption spectrum for typical plant pigments

What is a spec? How does it work? Review the content below.

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Spectophotometry uses these principles of light and color to help identify concentrations of various components in a solution. ​ Inside a spectrometer (or more specifically, a photospectrometer) a beam of light is shot a condenser and spectrum, then through the sample that contains a colormetric chemical indicator (an indicator that changes color, like the Benedict's solution we used in Unit 1). Subjectively we might have been able to say, "this color is darker so there must be more glucose." But if want an objective measurement of the "color" we can get the absorbance value from a spectrometer. A lower absorbance value means a darker color. 

When a color-sensitive indicator is used the absorbance values are directly proportional to the concentration in the solution.

• High Concentration = Dark Color = High Absorbance Values
• Low Concentration = Lighter Color = Low Absorbance Values

Spectrometers provide the absorbance values for a given sample by passing a specified wavelength through the sample at a constant distance (see below).

Click here for a review on spectrometry.

What will we do in lab?

How will we measure the rate of photosynthesis? ...By measuring the NADPH produced by chloroplasts in the light reactions! We will test active chloroplasts in different lighting environments to see how quickly the light reactions of photosynthesis occur. 

​Review the following steps to understand how we will quantify the photosynthetic rate. Use the links provided. You MUST understand the concepts below.

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Click to review lab equipment

• You will be provided with a solution of active chloroplast. The extraction process will be completed before lab. It's delicate and time intensive!
• You will create several environments, in test tubes, in which your chloroplasts can proceed through the process of photosynthesis (view example). What type of environment is up to your class (different types or colors of light).
• You will run your test for 5 minutes in these standard conditions with chloroplasts: 5 mL of tricine buffer + 5 mL DIP + 500 uL cold chloroplasts
• What the heck is DIP? Normally, the light reactions require NADP+. Photons excite electrons and they are used to reduced NADP+ to NADPH (which is then used in the Calvin Cycle). Instead of adding NADP to "feed" our chloroplasts, we are adding DIP as a stand in. [DIP = NADP+ and DIPH = NADPH]
• This is because DIP is color reactive, meaning we can use color changes to determine how much DIP is reduced to DIPH. That is how we will determine the rate of the light reactions in our differing environments: DIP is a blue/purple color, as it is reduced to DIPH the color intensity is diminished, it gets closer to clear. 
• DIP is a dark purple color. As the light reactions of photosynthesis proceed, the DIP becomes lighter and lighter. We will use the intensity of the DIP in our samples, as measured by a spectrophotometer, to determine the rate of photosynthesis. Review the flow chart below.
• So...after your experiments run in the test tubes, we will use the spectrometer to determine the absorbance value of the solution.  View the relationship between absorbance values and rate of the light reaction below

We will use these two devices in lab.

This is the "spec." Samples are placed in rectangular receptacles called cuvettes and inserted in the box to be "read."

This is a "lab quest mini." This is where we program the spec and also where we can read the results.

​If you feel confident with this material, click the bridge icon and navigate to Blackboard to take the LABridge. Be sure your Lab 7 Notebook Guide is ready to submit!

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Click here to get to WKU's blackboard to take your LABridge for this week. You will not be submitting a Lab Notebook Entry for this LABridge.

Lab 8: Protocol

In today's lab you will work with your lab group to develop a research proposal, conduct your experiment and begin to analyze your data. 

Exercise I. Develop a research proposal 

Exercise II. Conduct your experiment

​Exercise III. Begin to analyze your data

Lab Objectives: Following today's lab, you should be able to...

• Exercise I
• Exercise II
• Exercise III Post-Lab

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Exercise I. Set up Your Spec 

​Gloves and goggles all day.
Chloroplasts must be kept on ice.
These reactions will continue to proceed. You must act quickly but carefully.
All DIP will go into the hazardous waste bucket on the back bench.

​Procedure
You will determine the effect of light type or color (as decided as a class) on photosynthetic rate rates via spectrometry using DIP as a stand-in for NADP.​
Your TA will divide you up and let you know which variable you will test (based on class choice). You can test 5 test tubes at a time and may be able to run two sets of trails depending time.

1. ​​Review your provided materials. You are provided with: ​Gloves & goggles, beakers with DIP and tricine buffer, a spectrophotometer and Veiner LabQuest II, cuvettes, test tubes, an ice bath, chloroplasts in solution, micropipetters and tips, and a hazardous waste bag (for the tips).
2. Identify the spec and LabQuest.
3. Prepare your spectrometer. 
4. Power on LabQuest 2.
5. Plug in to outlet and spectrophotometer. Wait 15 seconds.
6. It should display an orange/red box with “USB:Abs.” If not, restart.
7. Tap orange screen with stylus and click “Calibrate.” Wait 90 seconds for lamp to warmup.
8. NOTE: Cuvettes are square and have two clear sides and two sides for labeling or gripping the cuvette. The cuvette should always be oriented in the V-Spec so the light passes through the clear sides (no ridges, sometimes marked with small triangle at the top).  Locate the light source in the V-Spec, and ask questions if you are not clear.  Always pay attention to this detail. Also, wipe the cuvettes off with a chem wipe before you begin. 
9. After warmup, place blank (DI water) cuvette into spectrophotometer. Make sure purple light passes through clear side of cuvette.
10. Tap “Finish Calibration” and “OK.”
11. Remove blank
12. Click on RED Absorbance rectangle >>> Change wavelength >>> get wavelength from your instructor (usually around 600nm). 

Take photos of your set-up and your experiment

Exercise II. Conduct Your Experiment

Procedure (Stadard Protocol)

1. You have been provided with a solution of active chloroplasts on ice.
2. The extraction process has already been completed. It's delicate and time intensive!
3. You will be able to run 5 trials at a time.
4. Label your test tubes by number.
5. Place 5 mLs of DIP in each experimental tube, to indicate the rate of the light reactions. 
6. Add 5 mLs of tricine buffer in each experimental tube, to keep a neutral environment for the chloroplasts.
7. Prepare your light source based on the variable you are testing.
8. Once you are ready to begin,  pipet 500 uL (0.5 mLs) cold chloroplast suspension into your tubes. Mix by tapping gently against palm.
9. Record the time in spreadsheet and illuminate.
10. The chloroplasts need to "work" for a minimum of 5 minutes. 
11. After your time limit has passed, quickly transfer 2 mLs from each cube into a correspondingly numbered cuvette.​
12. Begin inserting your tubes one at a time and reading/recording the absorbance value from each tube.
13. You must be swift. The reactions will continue to proceed. ​

Excel data sheet for Lab 8: Photosynthesis. Click to download.

Method to isolate functioning chloroplasts. Click to enlarge.

I still don't understand how this works!

• What the heck is DIP? Normally, the light reactions require NADP+. Photons excite electrons and they are used to reduced NADP+ to NADPH (which is then used in the Calvin Cycle). Instead of adding NADP to "feed" our chloroplasts, we are adding DIP as a stand in. [DIP = NADP+ and DIPH = NADPH]
• This is because DIP is color reactive, meaning we can use color changes to determine how much DIP is reduced to DIPH. That is how we will determine the rate of the light reactions in our differing environments: DIP is a blue/purple color, as it is reduced to DIPH the color intensity is diminished, it gets closer to clear. 
• DIP is a dark purple color. As the light reactions of photosynthesis proceed, the DIP becomes lighter and lighter. We will use the intensity of the DIP in our samples, as measured by a spectrophotometer, to determine the rate of photosynthesis. Review the flow chart below.
• So...after your experiments run in the test tubes, we will use the spectrometer to determine the absorbance value of the solution.  View the relationship between absorbance values and rate of the light reaction below​​

Click here for a review on photosynthesis.

Exercise III. Analysis

Procedure

1. You are ready to move forward. Now, what type of statistics should we use to determine if the mean respiration rate between our 2 groups is significantly different? ...hopefully this is an easy answer. Are the mean umols carbonic acid (H2CO3) produced in your two groups the same or are they different?
2. Use the tools below to complete your Lab Notebook Guide. 
3. The demo below will take you through, step-by-step.
4. You will need to create a table in Excel, a graph in Excel, run a T-test and answer a set of questions.
5. Be sure everyone has an updated copy of your excel spreadsheet and the Lab Notebook Guide to upload it in the next LABridge.

T-Test & bar graph demo (labs 7 & 8)

T-test Online Calculator.

Lab 8 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
BioSkillls 2: Reading & Making Graphs
BioSkillls 3: Interpreting Standard Error and Using Statistical Tests
BioSkillls 4: Working with Probabilities
Chapter 10: Photosynthesis

Faculty Spotlight: Dr. Chandra Emani

[email protected]

If you are especially curious about plants, Dr. Emani's research may interest you! He is investigating the use of molecular compounds produced by plants to meet human health needs, including the use of basil to produce anti-cancerous compounds.  He is also researching the use of sorghum and tobacco as biofuels. Dr. Emani is also exploring the ways plants can be used to "bio-clean" toxic areas (a process called, phytoremediation) using pine and sorghum to break down oil waste material near oil fields to reclaim the land and aid in restoration efforts (check out this cool video).

​​​Recent Publications
Chandrakanth Emani (Ed). (Mar 2018) The biology of plant-insect interactions: a compendium for plant biotechnologists. Science Publishers/CRC Press, Taylor & Francis Group: FL 304pp, ISBN 9781498709736 2. [BOOK]