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Bring Inquiry into Your Classroom

Bring Inquiry into Your Classroom. The 20 Question Approach. What is Inquiry?.

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Bring Inquiry into Your Classroom

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  1. Bring Inquiry into Your Classroom The 20 Question Approach

  2. What is Inquiry? • "the diverse ways in which scientists study the natural world and propose explanations based on the evidence derived from their work. Scientific inquiry also refers to the activities through which students develop knowledge and understanding of scientific ideas, as well as an understanding of how scientists study the natural world. - National Science Education Standards

  3. Why bother with inquiry? • Allows students to learn by doing an activity that they picked and designed • Relevance • Mimics “real” science • Allows for collaborative learning • Is a good way to link labs together (extensions, PBL)

  4. Steps to inquiry • What do you already know about it? • What kinds of questions can you ask? • Decide which questions: • can be answered by research / expert? • which are the “big” overarching questions? • and which ones would make good investigations?

  5. pGLO • What do we already know?

  6. What is a Plasmid? • Circular DNA – originally evolved by bacteria • Self-replicating • Has just a couple of genes • Must have an origin of replication (ori)

  7. pGLO Plasmid • Beta Lactamase (bla) • Ampicillinresistance • Green Fluorescent Protein (GFP) • Aequoreavictoria jellyfish gene • araC regulator protein • Regulates GFP transcription. If arabinose is present, then GFP is transcribed.

  8. Transformation Procedure

  9. What is the point of . . . . ? • Calcium Chloride in transformation solution: Ca+2 shields negative charge of DNA phosphates • Ice incubation: slows fluid cell membrane • 3. Heat-shock: increases permeability of membranes • 4. Nutrient broth incubation: • Allows beta-lactamase • expression Ca+2 heat

  10. RNA Pol Regulation of GFP – no arabinose • Without arabinose: • AraC is inactive • RNA pol doesn’t bind and transcribe GFP • (bla and araC are still transcribed) AraC Beta-lactamase

  11. RNA Pol Regulation of GFP – with arabinose • With arabinose: • AraC is active and helps RNA pol bind • RNA pol transcribes GFP • GFP is produced – bacteria glow

  12. pGLO Results

  13. pGLO • What kinds of questions can we ask?

  14. pGLO • More?

  15. Inquiry – leading questions • What are some good “starters” for investigation questions?

  16. Student Plan for Inquiry Investigation Question Research Independent, Dependent, Controlled variables Hypothesis Materials / equipment needed

  17. Student Plan - continued Procedure Data (how will it be collected) Analysis Results Would I do anything differently to improve the experiment? What new questions came up during the experiment?

  18. Teacher Plan for Experiment • Time needed • Earlier prep? Students? You? • Order extra supplies? • Materials needed • Need extras? • Need anything different / additional? • Equipment needed • Different temp water baths / incubators?

  19. Levels of Inquiry - Bio-Rad style • Level 1 Questions • simple to adapt • do not add extra days • Level 2 Questions • may add a few days onto the lab • may require a few additional materials to complete. • Level 3 Question • for students seeking a real challenge • will require additional days, techniques, and materials to answer. Less Time Student knowledge Materials Equipment More

  20. pGLO Inquiry – Level 1

  21. Higher Level Inquiry

  22. Inquiry in Action Amy Inselberger and Shari Cohen with student volunteers from Stevenson HS

  23. How does heat shock affect the competency of E. coli bacteria? Kept non-heat shock bacteria on ice during 50 second time other bacteria were heat shocked 80 colonies 0 colonies Bacterial lawn • RESULTS: • +pGLO bacterial plates that didn’t undergo heat shock have bacteria growth • TRANSFORMATION EFFICIENCY: • Heat shock +pGLO LB/amp/ara = 1116 • No Heat shock +pGLO LB/amp/ara = 414 • RESULTS: • -pGLO LB plate has bacteria growth & –pGLO LB/amp plate lacks growth • CONCLUSION: • Bacteria survived heat shock and are not resistant to ampicillin without plasmid genes Bacterial lawn 65 colonies

  24. How does heat shock affect the competency of E. coli bacteria? Kept non-heat shock bacteria on ice during 50 second time other bacteria were heat shocked • RESULTS: • Both +pGLO LB/amp/ara plates (heat shock & no heat shock) colonies glow green under UV light 65 colonies 175 colonies • CONCLUSIONS: • Heat shock increases the transformation efficiency & competency of E. coli • Bacteria can be transformed without heat shock

  25. Finding the time for inquiry • “Guide on the side vs. sage on the stage” • Optional lecture for students who need it (small group) • In-school field trip – in the lab all day • Flipped classroom

  26. Flipped Classroom Ideas • Create your own lecture / pre-lab library • Have students do the same – upload to YouTube, blog • Edmodo • YouTube • Bio-Rad technique videos bit.ly/b-rtechniques • Other multimedia • infographics • News articles • TV shows

  27. Will the presence of the mutagen silver nitrate (AgNO3), in three different concentrations mixed into the agar medium, alter gene expression in transformed E. coli cells? Approximately 5% of plate with 500mL of AgNO3 is covered with bacteria. Isolated colonies glow green under UV light Approimately95% of plate with 50mL of AgNO3 is covered with bacteria and cells glow green under UV light Approximately 50% of plate with 100mL of AgNO3 is covered with bacteria. Colonies glow green under UV light

  28. Will the presence of the mutagen silver nitrate (AgNO3), in three different concentrations in the agar medium, alter gene expression in transformed E. coli cells? • CONCLUSIONS: • The mutagen did not mutate the plasmid DNA since bacteria colonies on all plates were are able to survive on medium containing ampicillin and glowed green under UV light despite the addition of different amounts of silver nitrate added to the LB/amp/ara medium. • The silver nitrate qualitatively affected the size, shape, and texture of bacteria colonies growing on the medium. Further research is needed to determine why the colonies showed different macroscopic phenotypes in presence of mutagen. • The greater the amount of silver nitrate mixed into the agar medium, the smaller the number of bacteria were present, so the silver nitrate was a bacteria growth inhibitor.

  29. What is a plasmid? • A circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance gene or be modified to express proteins of interest

  30. pGlo Plasmid • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein (GFP) • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription

  31. What is Transformation? E. coli cell • Uptake of foreign DNA, often a circular plasmid

  32. Transformation Procedure Overview Day 2 Day 1 10

  33. Transformation Procedure • Suspend bacterial colonies in transformation solution • Add pGLO plasmid DNA • Place tubes on ice • Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates

  34. Transformation Procedure 7. Label plates as shown below (write on the bottom of the plates, not the lid). Add your initials to each plate. Save your tape!

  35. Transformation E. coli • Transformation solution of CaCl2. Ca+2 shields negative charge of DNA phosphates • Incubate on ice • slows fluid cell membrane • 3. Heat-shock • Increases permeability of membranes • 4. Nutrient broth incubation • Allows beta-lactamase • expression Ca+2 heat

  36. Methods of Transformation • Electroporation • Electrical shock makes cell membranes permeable to DNA • Calcium Chloride/Heat-Shock • Chemically-competent cells uptake DNA after heat shock

  37. Why perform each transformation step? • Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH

  38. Why perform each transformation step? E. coli 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression

  39. Transformation Procedure Heat shock 8. Carefully take your ice cup to the water bath. Heat shock cells by placing the float in the water bath for 50 seconds Return to ice for 2 minutes

  40. Volume Measurement

  41. Transformation Procedure Recovery 9. Add 250ul of LB to each tube. Leave at room temperature for 10 minutes

  42. What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins

  43. Grow?Glow? • On which plates will colonies grow? • Which colonies will glow?

  44. Student Inquiry Questions to consider: • How important is each step in the lab protocol? • What part of the protocol can I manipulate to see a change in the results? • Ampicillin concentration • Arabinose concentration / timing • Heat shock temperature or time • Time on ice before and after heat shock • Amount of plasmid • Amount of bacteria • Phase of bacteria used for transformation • How do I insure the change I make is what actually affected the outcome? • Importance of controlling other variables • Collaborative approach / share data • Write protocol, get approval, and do it!

  45. StudentInquiry More Advanced Questions • Are satellite colonies also transformed? • What other genes might the pGLO plasmid contain? • Can I map the plasmid? • Can I remove the pGLO gene? • Can I remove the regulation of GFP so I don’t need to add arabinose? • Can I detect the presence of the GFP gene using PCR?

  46. Student InquiryTeacher Considerations • What materials and equipment do I have on hand, and what will I need to order? • Extra plates, LB, agar, plasmid, ampicillin, arabinose? • Incubator, water bath (different temps) • Other supplies depending on student questions • Consider buying extras in bulk or as refills – many have 1 year + shelf life. • What additional prep work will I need? • Order supplies • Pour plates (different media? different amounts?) • Make starter plates (will you need transformed bacteria?) • How much time do I want to allow? • Limited time? Have students read lab and come up with inquiry questions and protocol before they start. Collaborative approach. • Will you need multiple lab periods? • Will everyone need the same amount of time?

  47. Transformation Procedure Plating Bacteria 10. Put 100 ul of solution onto the appropriate plates 11. Streak plates 12. Stack / tape plates and place in incubator New pipet! New loop!

  48. Green Fluorescent Protein (GFP) Chromatography Kit GFP Purification Kit Advantages • Cloning in action • Links to biomanufacturing • Biopharmaceutical development • Amazing visual results

  49. SDS PAGE Extension

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