Bring Inquiry into Your Classroom The 20 Question Approach

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

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)

What do you already know about it? 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?

pGLO What do we already know?

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)

pGLO Plasmid Beta Lactamase (bla) Ampicillin resistance Green Fluorescent Protein (GFP) Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription. If arabinose is present, then GFP is transcribed.

Transformation Procedure

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

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 RNA Pol Beta-lactamase

Regulation of GFP – with arabinose AraC is active and helps RNA pol bind RNA pol transcribes GFP GFP is produced – bacteria glow RNA Pol

pGLO Results

What kinds of questions can we ask? pGLO What kinds of questions can we ask? Question Expert? Big idea? Investigation? What if we skipped heat shock? I What if we didn’t use CaCl2? KCl What happens if we change time on ice bath / heat shock? What happens if you change the temperature?

More? pGLO Question Expert? Big idea? Investigation? Which plate should have growth / glow? What happens if we don’t use LB/Amp plates? Why do we want to add plasmids (DNA) to bacteria? Relevance? Big idea! How did scientists develop technique? Research What happens if you skip recovery? Investigation

Inquiry – leading questions What are some good “starters” for investigation questions? What will happen if I? What are the effects of ... Will it ... if I do ... How will... affect ... How many ... How much ... How does it ... (any sense except taste)

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

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?

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?

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

pGLO Inquiry – Level 1

Higher Level Inquiry

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

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 Bacterial lawn 0 colonies 65 colonies Bacterial lawn 80 colonies 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

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 65 colonies 175 colonies RESULTS: Both +pGLO LB/amp/ara plates (heat shock & no heat shock) colonies glow green under UV light CONCLUSIONS: Heat shock increases the transformation efficiency & competency of E. coli Bacteria can be transformed without heat shock

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

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

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

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.

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

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

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

Transformation Procedure Overview Day 1 10 Day 2

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

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!

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

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

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

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

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

Volume Measurement

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

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

Grow? Glow? On which plates will colonies grow? Which colonies will glow?

Questions to consider: How important is each step in the lab protocol? 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!

Student Inquiry 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?

Student Inquiry Teacher 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?

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

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

SDS PAGE Extension

Webinars Enzyme Kinetics — A Biofuels Case Study Real-Time PCR — What You Need To Know and Why You Should Teach It! Proteins — Where DNA Takes on Form and Function From plants to sequence: a six week college biology lab course From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars

pGLO Plasmid pGLO Plasmid DNA