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Adapting labs for inquiry

Adapting labs for inquiry. EDS 198 . Agenda. Lab Review and Analysis of Results Adapting Lab for Inquiry Protein Purification Lab Background. Review. What was the purpose of the lab last week? How did we accomplish this?.

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Adapting labs for inquiry

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  1. Adapting labs for inquiry EDS 198

  2. Agenda • Lab Review and Analysis of Results • Adapting Lab for Inquiry • Protein Purification Lab Background

  3. Review • What was the purpose of the lab last week? • How did we accomplish this? The purpose of the lab was to change the genotype of e. coli to change it’s phenotype.

  4. Review: How We Make E.coli Glow Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days DNA  RNA Protein Bacteria now express cloned fluorescent protein…

  5. Review: 3 agar Plates What was the purpose of growing bacteria on each plate? LB/AMP + LB/No Amp - LB/AMP - On the post lab section of your protocol, draw out what you expect to see growing on each plate.

  6. Analyze: let’s see Your Plates Demo using a black light Questions for you and your students to think about: What do you observe? Was it what you expected? Why or why not? What do you think happened to cause this to occur?

  7. Side note: Satellite colonies Satellite Colony: small bacterial colonies that can surround a large antibiotic-resistant colony Why satellite colonies occur: antibiotic resistant bacteria secrete beta-lacatamase to break down ampicillin in the surrounding area allowing non-resistant bacteria to grow in that zone.

  8. Protein Purification background • What is a protein? • A large molecule composed of chains of amino acids

  9. Protein purification Background What do proteins do? Hormones Hair and Nails Muscle Contraction Structural Support Receptors, membrane channels Enzymes Antibodies Nutrient Storage

  10. Protein purification Background What do proteins do? • Inner Life of a Cell - http://www.youtube.com/watch?v=wJyUtbn0O5Y • Myasthenia Gravis –

  11. Protein purification Background How do you make a protein? DNA mRNA Protein Trait

  12. Protein Purification Background • Why would you ever want to purify a protein? • To study the protein • To make medicine – insulin, lactase

  13. Protein Purification Background Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

  14. How would you Purify a Protein • Things to consider: • There are thousands of different proteins in a cell • The characteristic of the protein you want to isolate

  15. General Steps of protein purification • Lyse (cut) open the cells. • Separate the cellular components by size • Distinguish protein of interest • Separate protein of interest • Retrieve protein of interest See whiteboard

  16. Protein purification protocol Steps • Collect Bacteria • Scrape transformed cells into tube with TE Buffer • Break Open Cells • Add lysozyme to weaken cell wall • Snap Freeze to blow up cell

  17. Side note: What is lysozyme Lysozyme a naturally occurring enzyme used to weaken the cell wall of e. coli through a hydrolysis reaction –Found in mucus, tear, egg whites

  18. Side-note: what is snap freeze • Snap Freeze – The process of quickly freezing something

  19. Protein purification protocol Steps 3. Centrifuge the cells to separate the heavy cell material from the lighter cell material supernatant pellet

  20. Protein purification protocol Steps 3c. Add elution buffer to column and collect solution in a new tube 3a. Mix supernatant with nickel beads 3b. Pass the supernatant and nickel bead mixture through the column and into a waste tube 3. Separate the fluorescent protein from other light cellular debris using column chromatography

  21. Side- Note: How do the nickel beads work? his-his-his-his-his-his His- tag: a chain of histidine amino acids

  22. Side-Note: How do the nickel beads work? The his-tag on the fluorescent protein and nickel bind like 2 magnets Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+

  23. Side-Note: How does the elution buffer work? Elution buffer contains a molecule called imidizole that has a stronger attraction to the nickel beads Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Video: www.youtube.com/watch?v=FUAQKjKT99Y

  24. Proteins purified! Finished! Now you have a pure sample containing only fluorescent proteins

  25. Making the lab inquiry based Using our understanding of inquiry, how can we incorporate these ideas to make the lab more inquiry based? 20 min brain storm Student Choice What is inquiry? What does Inquiry mean to you? Topic is relevant and meaningful Students create their own understanding

  26. Making the lab inquiry based Share out how to make lab inquiry based What inquiry is: Student Choice Students create their own understanding Topic is relevant and meaningful ___________________ • Ask students to come up with their own way of purifying protein • Ask students to figure out why each step is needed. (What is happening at the molecular level?) • Ask students what they would use with this technology. (Ethics discussion) • Do lab twice and change a variable. (we have 7 sets total)

  27. Lesson Planning for next week Agenda for Next Week: • Look at Results of Bacterial Transformation with Students • Random Group of Students? • Start Protein Purification • Incorporate inquiry based techniques • Create a lesson plan • Determine your own pacing for your own group • Each group will get at least 2 kits • Clean up and Snacks

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