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Download Lisa Byers UNIT 6: Genetic Transformations Unit Plan
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NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 1 of 5 Lisa Byers UNIT 6: Genetic Transformations Unit Plan Unit Outline: Lesson 1: Introduction to Transformation and lab set up Day 1 - Title of Lesson: pGLO Transformation Day 1 Time: One period or 55 minutes. Prior Knowledge Necessary: DNA’s role in the continuity of an organism, the process of analyzing DNA through gel electrophoresis, and basic understanding of heredity (genotypes vs. phenotypes) are all important concepts that should be covered before this activity. General Concepts: Bacterial DNA can be transformed through use of a plasmid. National Science Education Standards Content Standard A: Science as Inquiry Abilities Necessary to do Inquiry NSES p.175 “Use technology and mathematics to improve investigations and communications” “Formulate and revise scientific explanations and models using logic and evidence” Content Standard C: Life Science The Cell NSES p.184 “Cells have particular structures that underlie their functions” “Cell functions are regulated which allows cells to respond to their environment and to control and coordinate cell growth and division” The Molecular Basis of Heredity NSES p.185 “In all organisms, the instructions for specifying the characteristics of the organism are carried in DNA . . . The structural and chemical properties of DNA explain how the genetic information is encoded in genes and replicated. Each DNA molecule in a cell forms a single chromosome.” The Interdependence of Organisms NSES p.186 “Organisms both cooperate and compete in ecosystems. The interrelationships and interdependencies of these organisms may generate ecosystems that are stable. . .” AZ High School State Standards Science Standards Strand 11: Concept 1: PO 4 Concept 2: PO 5 Strand 2: Concept 1: PO 1 Strand 4: Concept 1: PO 1 & PO 2 Concept 2: PO 1 & PO 2 Concept 3: PO 2 NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 2 of 5 Common Misconceptions: « Prior beliefs were found to be the major influence in a pupil’s understanding of the experiments that use sterile control plates in a bacteriological experiment (and) could not be understood if a child’s concepts of living, size, growth, and reproduction in bacteria are not understood » (Driver, etc al. Pg. 57). Essential Questions: - Describe how you could use two LB/agar plates, some E. coli and ampicillin to determine how E. coli cells are affected by ampicillin. - What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells? - What other process have we just learned about that we know can utilize gene therapy? - What would the gene code for? - On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your prediction. - If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. - Which plates should be compared to determine if any genetic transformation has occurred? Why? - What is meant by a control plate? What purpose does a control plate serve? - What else does the plasmid need besides the DNA of the coding enzyme? Behavioral Objectives: The students will be able to safely administer all protocol for the laboratory. The students will be able to correctly answer the essential questions. The students will be able to describe the role of LB, ampicillin, Arabinose, and pGLO plasmid. Description of the Lab: This activity is a laboratory activity from BIO-Rad pGLO transformation kit. The students will be setting up a transformation of DNA to compare with the process and production of Chymosin. Incorporation of Inquiry: The activity is a strict procedure that needs to be followed for the intended outcome to happen. It is on the low end of the inquiry scale, much closer to direct instruction. Materials: pGLO Student manual E. coli starter plate Poured agar plates Transformation solution LB nutrient broth Inoculation loops Pipets Foam microtube holder/float Container full of crushed ice Marking pen Rehydrated pGLO plasmid 42 C water bath and thermometer 37 C incubator NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 3 of 5 Safety: Students have a safety contract as agreed to at the beginning of the year. The teacher will review sterile technique to prevent contamination of lab surfaces and safety. In the contract students promise to report inappropriate behavior to the instructor immediately, promise not to rough house in the lab, promise to dress appropriately to lab, promise to not eat or drink anything while in the lab, promise to know what they are going to do before they conduct a lab. Textbook: The diagram and background information comes from Biotechnology Laboratory Manual – Ellyn Daugherty Biotechnology Explorer pGLO Bacterial Transformation Kit instructors guide – Bio-Rad Biology, Principals and Explorations – Holt, Rineheart, and Winston Unifying concepts: Make biotechnology relevant to the students and tie in how it relates to the science they have already learned. Discover how scientific processes can be used for many different purposes. Visually seeing how DNA goes to RNA, which then is turned into a protein that is expressed a trait. Assessments: The students will accurately complete all essential questions including a ticket out the door as a reflection and assessment. Real world connections: Bacterial transformation techniques are used in the field of Biotechnology. An example of bacterial transformations is the process by which Chymosin is created. Lesson 2: Collection and interpretation of data Warm up – As students enter they are to answer the following question from their homework from the previous night: Which is the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse? Describe your reasoning. 5 min - The students will receive their plates to complete Consideration 2 (pg 30 in student manual). - Describe how you could use two LB/agar plates, E. coli and some ampicillin to determine how E. coli cells are affected by ampicillin. - What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells? - What other process have we just learned about that we know can utilize gene therapy? 5 min - The teacher will randomly call on students for their answers. The teacher will probe for students to clarify misunderstanding. 5 min – A brief lecture and review of the visual act of transformation (Consideration 3 pg 31). 30 min - The students are to follow the protocol listed below (taken from pGLO Transformation teacher guide pages 14-15): NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 4 of 5 1. Label one closed micro test tube +pGLO and another –pGLO. Label both tubes with your group name. Place them in the foam tube rack. 2. Open the tubes and using a sterile transfer pipette, transfer 250 micro-liters if transformation solution (CaCl2) 3. Place the tubes on ice. 4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the –pGLO tube. 5. Examine the pGLO plasmid DNA solution with the UV lamb. Note your observations. Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a loop full. There should be a film of plasmid solution across the rind. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loop full into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. Also close the –pGLO tube. Do no add plasmid DNA to the –pGLO tube. Why not? 6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottom of the tubes sticks out and make contact with the ice. 7. While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as follows: - LB/amp plate: +pGLO - LB/amp/ara plate: +pGLO - LB/amp plate: -pGLO - LB plate: -pGLO 8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the change from the ice (0° C) to 42° C and then back to the ice must be rapid. Incubate on ice for 2 min. 9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipette, add 250 micro-liters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipette for the other tube. Incubate the tubes for 10 minutes at room temperature. 10. Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100 micro-liters of the transformation and control suspensions onto the appropriate plates. 11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface being careful not to gouge the surface. 12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack upside down in the 37° C incubator until the next day. 5 min – Students clean up and may begin to work on their homework. 5 min - Ticket out the door: What would the gene code be for if we did this experiment to help make cheese production more efficient? Answer: the enzyme Chymosin NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 5 of 5 Homework: - On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your prediction. - If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. - Which plates should be compared to determine if any genetic transformation has occurred? Why? - What is meant by a control plate? What purpose does a control plate serve? - What else does the plasmid need besides the DNA of the coding enzyme? NAU BIOTECH © 2009 Lisa Byers