You Are What You Eat: An Investigation of Macromolecules

You Are What You Eat: An Investigation of Macromolecules Teacher Materials Students will use standard indicators to test carbohydrates, lipids, proteins, and nucleic acids. Learning Goals, Objectives, and Skills... 2 Instructor Planning Guide... 3 Instructor Preparation Guide... 5 Answers to Student Questions... 9 Standard Alignments.10 Calculation Tool for Ordering NEB Reagents.12 Last updated: 10/15/18

You Are What You Eat Learning Goals Student Learning Goals: Students will understand the basic structures and functions of the four classes of macromolecules. Students will understand that most macromolecules are polymers. Students will understand how hydrolysis and dehydration synthesis reactions relate to macromolecules. Students will understand that nearly all food is derived from organisms, and therefore is made up of cells. Students will understand that cells contain carbohydrates, lipids, nucleic acids, and proteins. Students will understand that the presence of macromolecules in cells can be detected with simple chemical tests. Student Learning Objectives: Students will relate the cycling of nutrients in the biosphere to the cycling of nutrients within organisms as macromolecules are broken down and synthesized. Students will prepare positive and negative controls for the tests that determine the presence of each of four macromolecules in two different solutions. Scientific Inquiry Skills: Students will design and conduct scientific investigations. Students will make measurements and record data. Students will compare their results to those of the control and to their predictions. Students will follow laboratory safety rules and regulations. Laboratory Technical Skills: Students will demonstrate proper use of micropipettes. 2

You Are What You Eat Instructor Planning Guide There are two approaches to this lab depending on your students and availability of lab space. The first option is to work through the manipulation of the models for all the macromolecules and then move onto the testing of the samples. The second option is to work with the models for one macromolecule and do the testing of the sample for that macromolecule before moving onto the second macromolecule. Experimental Timing Tips: From start to finish this lab takes approximately 120 minutes. However, there are many good stopping points in this protocol that make it possible to complete the lab in a series of 45-50 minute periods. We recommend that pre-lab discussion and any demonstrations of the procedures take place the day before the lab. Testing for each macromolecule should take 10-20 minutes, depending on the sample The first macromolecule generally takes longer than the others since the students are learning how to manipulate the models and set up the testing. Specialized Equipment: p200 micropipettes (or graduated disposable pipettes) hot plate or microwave UV or blue LED light Ordering information: Indicators, glucose (dextrose) and BSA samples were ordered from Fisher Scientific. Oil and spray starch were purchased at the grocery store. The nucleic acid is lambda DNA and can be ordered from New England BioLabs (at no cost) by going to their website (https://www.neb.com). A calculation tool for ordering NEB Reagents for this lab can be found on the final page of this document. Procedure Tips: 1. Before starting the experiment, ask students to check their materials list to make sure they have everything they will need for that part of the experiment. 2. Make sure students understand safety procedures and know the location of safety equipment. 3. Make sure students know how to use the transfer pipettes to measure the specific volumes. A black line drawing of the pipette with the graduations is a helpful tool for students to have as a reference (see Appendix 1). 4. Each type of macromolecule is tested separately so the lab procedure can easily be spread over several days. However, the lab can be completed in one long block. 3

5. The water for Benedict s testing does not have to be boiling. Just make sure it is at least 90 C. It can be dispensed to the students in the 250 ml beakers. 6. The amount of reagent listed in the set-up is for 15 lab groups. If running this lab with multiple classes, you might want to increase the amounts so you don t have to refill the containers. 7. Iodine should be stored in light blocking bottles or in the dark. 8. Keep in mind that the DNA concentration is listed as g or micrograms for the concentrated DNA and ng or nanograms for the diluted DNA. 9. Make sure students understand that they are looking for the color change of the indicator. Teaching Tips: Alternate working with models of each type of macromolecule with testing the macromolecule. For example, have students manipulate models of the carbohydrates and then do the testing. If using this approach, the lab can be spread over several days. Modeling Organic Molecules (https://www.massbioed.org/educators/curriculum/7-you-are-what-you-eat-investigatingmacromolecules ) walks students through an exploration of macromolecules using Cell Zone Molecular Puzzles (https://www.cellzone.org/products/molecular-puzzles/ ). Once students have learned how to set up the tests and seen the results from the standards they could test some unknowns such as egg white (protein), apple juice (glucose), potato (starch), melted butter (lipid). Safety Considerations: Gloves, lab coats and eye protection should be used whenever possible as part of good laboratory practice. Practice sterile techniques whenever possible, to avoid contamination of reagents. Exercise caution when heating or distributing the hot water. Exercise caution when working with electrical equipment. Always wash hands thoroughly after handling biological materials or reagents. Obtain the Material Safety Data Sheets (MSDS) available from the suppliers, and follow all safety precautions and disposal directions as described in the MSDS. Check with your school s lab safety coordinator about proper disposal of all reagents and solutions containing DNA stains. 4

You Are What You Eat Instructor Preparation Guide Materials: This guide assumes 30 students, working in groups of two, for a total of 15 groups. Materials for Advanced Teacher Preparation: 0.2 g BSA (Fisher Catalog #50-753-3052) 2 g glucose (dextrose) (Fisher Catalog #S25295B) 5 g cornstarch spray starch 45 ml vegetable oil 1 tube lambda DNA at 500 g/ml (NEB# N3011S) 1 tube will have 250 g DNA in 500 L solution 45 ml Benedict s solution (Fisher Catalog #S25193A) 45 ml Biruet solution (Fisher Catalog #S25200B) 45 ml Lugol s solution (iodine) (Fisher Catalog #IS5502) 45 ml Sudan IV solution (Fisher Catalog #S25592) 20 L GelGreen solution (10,000X) (Fisher Catalog #EC1995) 350 ml distilled water 1 100 ml graduated cylinder 1 p20 micropipette and tips 1 p200 micropipette and tips 1 p1000 micropipette and tips 1 10 ml graduated pipette or graduated cylinders 3 100 ml flasks or bottles 110 15 ml conical tubes or small bottles with caps 15 15 ml amber conical tubes or small bottles with caps (Fisher Catalog #03-395-119) 35 microcentrifuge tubes (1.5 ml or 2.0 ml) 3-5 conical tube racks (optional) 1 microcentrifuge tube racks 1 permanent marker 1 balance Caution: Lugol s solution (iodine) is light sensitive and should be stored in amber container. Caution: GelGreen is light sensitive and should be stored in the dark 5

Student Workstation: Common Workstation: 3 ml Biuret solution Microwave or hot plate 3 ml Benedict s solution UV or blue light 3 ml Lugol s solution 600 ml beaker of water 3 ml Sudan IV solution 60 μl GelGreen solution (1X) 3 ml BSA solution (2 mg/ml) 3 ml glucose solution (2%) 3 ml Starch solution (5%) 3 ml vegetable oil 30 μl lambda DNA (200 ng/ μl) 20 ml distilled H 2O 1 p200 micropipette and tips 10 1 ml graduate pipettes 2 microcentrifuge tubes (1.5 ml or 2.0 ml) 8 test tubes 1 test tube rack 1 microcentrifuge rack 1 test tube holder 1 250 ml beaker 1 permanent marker 1 waste container Easy Substitutions: Purchase salmon sperm DNA (Fisher Scientific NC 9385184). The DNA (20 mg) comes lyophilized and looks like a wad of cotton. Resuspend in 20 ml of sterile water or TE. It will take a while to go into solution you might have to vortex it. Once resuspended, aliquot 1 ml into 20 sterile microcentrifuge tubes. Store at -20 C until needed. At -20 C it will have an almost indefinite shelf life. Depending on the size of your school this should last several years. Spray starch is an easy alternative to mixing up a starch solution. Two or three squirts into 200 ml of water should be sufficient. Also cornstarch dissolves in water more readily than soluble starch. Egg white can be substituted for BSA. Set-up Calendar: 2 weeks before lab Check supplies and order any needed materials. If making any substitutions to the supply list, edit the student protocol accordingly. 1 day before lab Set up student lab stations with all durable materials according the materials listed above. 6

Prepare BSA solution 1. Add 0.2 g BSA to 100 ml distilled water and mix well 2. Aliquot 3 ml into small bottles with caps. Prepare one bottle per lab group. 3. Store at 4 C until morning of use. Dispense 3 ml Biuret solution into small bottles. Prepare one bottle per lab group. Prepare 2% glucose solution 1. Add 2 g glucose to 100 ml of water and mix well 2. Aliquot 3 ml into small bottles with caps. Prepare one bottle per lab group. Prepare starch solution 1. Add 5 g cornstarch to 100 ml of water and mix well. An easy alternative is to use spray starch squirt enough starch into the water until it turns milky. 2. Starch solution should be tested with iodine to make sure it is concentrated enough to give a clear result 3. Aliquot 3 ml of starch solution into small bottles with caps. Prepare one bottle per lab group. 4. Starch solution should be stored at 4 C until the morning of use. (At room temperature bacteria can break the starch down) Dispense 3 ml of Benedict s solution into small bottles. Prepare one bottle per lab group. Dispense 3 ml of iodine into small bottles. Prepare one bottle per lab group. Caution: Lugol s solution (iodine) is light sensitive and should be stored in amber container. Prepare BSA solution 1. Add 0.2 g BSA to 100 ml water and mix well 2. Aliquot 3 ml into small bottles with caps. Prepare one bottle per lab group. 3. Store at 4 C until morning of use. Dispense 3 ml Biuret solution into small bottles. Prepare one bottle per lab group. Dispense 3 ml of vegetable oil into small bottles. Prepare one bottle per lab group. Dispense 2 ml Sudan IV into small bottles. Prepare one bottle per lab group. Prepare Gel Green (or other DNA Stain) dilution 1. Add 1980 L sterile dh 2O to a 15 ml conical tube 2. Add 20 L Gel Green and swirl to mix 3. Aliquot 60 L into microcentrifuge tubes. Prepare one tube per lab group. Caution: GelGreen is light sensitive and should be stored in the dark. Prepare DNA Solution 1. Dilute lambda DNA to 200 ng/ml 2. If using NEB lambda (#N3011S) add 200 L of 500 g/ml lambda DNA to 300 L sterile dh 2O and mix 3. Aliquot 30 L into microcentrifuge tubes 7

Morning of lab Set up student lab stations remaining reagents according to materials list. Set up common station During the Lab Benedict s test: Heat water in microwave or on hot plate as students are preparing their samples. Dispense into beakers or cups and distribute. 8

You Are What You Eat Answers to Student Questions Protocol-Embedded: p3. Lactose is C 12H 22O 11 p4. Answer will vary but may include: Proteins act enzymes, as antibodies, as structural components, for cell signaling p4. cellulose is a carbohydrate (polysaccharide) p5. water and lipids don t mix because lipids are nonpolar so they don t dissolve in water or watery solution Pre-Lab: 1. carbohydrates, proteins, lipids, and nucleic acids 2. omelet with black beans and cheese all of these foods are good sources of protein. Bread is primarily a carbohydrate, butter is primarily a lipid, and jam is primarily made up of carbohydrates. 3. Carbons-6; hydrogens- 12; oxygens-6 Glucose (monosaccharide) Dehydration synthesis 5. 1 a; 2 e; 3 b; 4 c; 5 d. Post-Lab and Analysis: 1. Starch because the iodine changed to black, protein because the Biuret changed to purple. There is not any DNA or glucose since neither indicator changed colors. 2. Strawberries are made up of cells, and all cells are surrounded by a phospholipid bilayer (cell membrane). So, strawberries definitely contain lipids! 9

You Are What You Eat Standards Alignments MA Science and Technology/Engineering Standards High School (2016) Biology HS-LS1-6. Construct an explanation based on evidence that organic molecules are primarily composed of six elements, where carbon, hydrogen, and oxygen atoms may combine with nitrogen, sulfur, and phosphorus to form monomers that can further combine to form large carbon-based macromolecules. NRC Practices Asking questions and defining problems Planning and carrying out investigations Analyzing data Mathematical and computational thinking Constructing explanations and designing solutions Engaging in argument from evidence Obtaining, evaluating and communicating information Next Generation Science Standards High School (2013) Life Sciences HS-LS1-6. Construct and revise an explanation based on evidence for how carbon, hydrogen, and oxygen from sugar molecules may combine with other elements to form amino acids and/or other large carbon-based molecules. Common Core State Standards Connections: ELA/Literacy RST.9-10.7 RST.9-10.8 RST.11-12.1 RST.11-12.7 Translate quantitative or technical information expressed in words in a text into visual form (e.g., a table or chart) and translate information expressed visually or mathematically (e.g., in an equation) into words. Assess the extent to which the reasoning and evidence in a text support the author s claim or a recommendation for solving a scientific or technical problem. Cite specific textual evidence to support analysis of science and technical texts, attending to important distinctions the author makes and to any gaps or inconsistencies in the account. Integrate and evaluate multiple sources of information presented in diverse formats and media (e.g., quantitative data, video, multimedia) in order to address a question or solve a problem. 10

RST.11-12.8 RST.11-12.9 WHST.9-12.1 WHST.9-12.2 WHST.9-12.5 WHST.9-12.7 WHST.9-12.9 SL.11-12.5 Mathematics MP.2 MP.4 HSF-BF.A.1 HSF-IF.C.7 HSN.Q.A.1 HSN.Q.A.2 HSN.Q.A.3 HSS-IC.A.1 HSS-IC.B.6 Evaluate the hypotheses, data, analysis and conclusions in a science or technical text, verifying the data when possible and corroborating or challenging conclusions with other sources of information. Synthesize information from a range of sources (e.g., texts, experiments, simulations) into a coherent understanding of a process, phenomenon, or concept, resolving conflicting information when possible. Write arguments focused on discipline-specific content. Write informative/explanatory texts, including the narration of historical events, scientific procedures/ experiments, or technical processes. Develop and strengthen writing as needed by planning, revising, editing, rewriting, or trying a new approach, focusing on addressing what is most significant for a specific purpose and audience. Conduct short as well as more sustained research projects to answer a question (including a self-generated question) or solve a problem; narrow or broaden the inquiry when appropriate; synthesize multiple sources on the subject, demonstrating understanding of the subject under investigation. Draw evidence from informational texts to support analysis, reflection, and research. Make strategic use of digital media (e.g., textual, graphical, audio, visual, and interactive elements) in presentations to enhance understanding of findings, reasoning, and evidence and to add interest. Reason abstractly and quantitatively. Model with mathematics. Write a function that describes a relationship between two quantities. Graph functions expressed symbolically and show key features of the graph, by hand in simple cases and using technology for more complicated cases. Use units as a way to understand problems and to guide the solution of multi-step problems; choose and interpret units consistently in formulas; choose and interpret the scale and the origin in graphs and data displays. Define appropriate quantities for the purpose of descriptive modeling. Choose a level of accuracy appropriate to limitations on measurement when reporting quantities. Understand statistics as a process for making inferences about population parameters based on a random sample from that population. Evaluate reports based on data. 11

You Are What You Eat Calculation tool for ordering NEB Reagents for: You Are What You Eat Please keep in mind that NEB is a fantastic and generous partner and will provide up to $1000 of reagents for each school. Please check with your colleagues to coordinate your ordering to ensure that your school plans ahead for ALL of the planned labs requiring NEB reagents, and please, only order as much as you need. The calculation tool below will help you determine how much of each reagent to order. Importantly, the amount needed per group shown below includes the extra needed in case of mistakes or when aliquots are provided for each group. Fill out the chart below to determine how many tubes of each of the reagents you need to order. The following are important to keep in mind: The number of groups will vary depending on your classes and equipment. Calculation tool: Example NEB Reagent NEB Catalog # Amount of Reagent In NEB Tube Amount Needed per Group Total Number of Groups Doing the Lab Total Amount You Will Need # Tubes Needed Reagent X X0000 40 L 4 L 8 32 L 1 You fill this in 4 L X (# groups) 32 L < 40 L NEB Reagent NEB Catalog # Amount of Reagent In NEB Tube Amount Needed per Group Total Number of Groups Doing the Lab Total Amount You Will Need # Tubes Needed Lambda DNA N3011S 500 L 1 L Once completed, you can submit your order here: https://www.neb.com/forms/bioteach 12

Appendix 1. Units of measure on 1 ml graduated transfer pipet 1 ml 750 μl 500 μl 250 μl 100 μl 13