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. 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?

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) Ampicillin resistance
Green Fluorescent Protein (GFP)
Aequorea victoria 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. 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

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

12. pGLO Results

13. 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?

14. 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

15. 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)

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
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

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
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

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.

30. 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

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

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

33. Transformation Procedure Overview
Day 1 10
Day 2

34. 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

35. 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!

36. 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

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

38. 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

39. 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

40. 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

41. Volume Measurement

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

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

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

45. 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!

46. 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?

47. 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?

48. 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!

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

50. SDS PAGE Extension

51. 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

52. pGLO Plasmid
pGLO
Plasmid DNA