bring inquiry into your classroom the 20 question approach
TRANSCRIPT
Bring Inquiry into Your ClassroomThe 20 Question Approach
Biotechnology Explorer™ | explorer.bio-rad.com2
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
Biotechnology Explorer™ | explorer.bio-rad.com3
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)
Biotechnology Explorer™ | explorer.bio-rad.com4
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?
Biotechnology Explorer™ | explorer.bio-rad.com5
pGLO
What do we already know?
Biotechnology Explorer™ | explorer.bio-rad.com6
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)
Biotechnology Explorer™ | explorer.bio-rad.com7
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.
Biotechnology Explorer™ | explorer.bio-rad.com8
Transformation Procedure
Biotechnology Explorer™ | explorer.bio-rad.com9
What is the point of . . . . ?
Ca+2
heat
1. Calcium Chloride in transformation solution: Ca+2
shields negative charge of DNA phosphates
2. Ice incubation: slows fluid cell membrane
3. Heat-shock: increases permeability of membranes
4. Nutrient broth incubation:Allows beta-lactamase expression
Biotechnology Explorer™ | explorer.bio-rad.com10
RNA Pol
Beta-lactamase
AraC
Regulation of GFP – no arabinose
Without arabinose:
•AraC is inactive
•RNA pol doesn’t bind and transcribe GFP
•(bla and araC are still transcribed)
Biotechnology Explorer™ | explorer.bio-rad.com11
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
Biotechnology Explorer™ | explorer.bio-rad.com12
pGLO Results
Biotechnology Explorer™ | explorer.bio-rad.com13
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 I
What happens if we change time on ice bath / heat shock?
I
What happens if you change the temperature? I
Biotechnology Explorer™ | explorer.bio-rad.com14
pGLO
More?
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
Biotechnology Explorer™ | explorer.bio-rad.com15
Inquiry – leading questions
What are some good “starters” for investigation questions?
Biotechnology Explorer™ | explorer.bio-rad.com16
Student Plan for Inquiry Investigation
Question
Research
Independent, Dependent, Controlled variables
Hypothesis
Materials / equipment needed
Biotechnology Explorer™ | explorer.bio-rad.com17
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?
Biotechnology Explorer™ | explorer.bio-rad.com18
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?
Biotechnology Explorer™ | explorer.bio-rad.com19
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
More
Time
Student knowledge
Materials
Equipment
Biotechnology Explorer™ | explorer.bio-rad.com20
pGLO Inquiry – Level 1
Biotechnology Explorer™ | explorer.bio-rad.com21
Higher Level Inquiry
Biotechnology Explorer™ | explorer.bio-rad.com22
Inquiry in Action
Amy Inselberger and Shari Cohen with student volunteers from Stevenson HS
Biotechnology Explorer™ | explorer.bio-rad.com23
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:•-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
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
65 colonies Bacterial lawn
80 coloniesBacterial
lawn0 colonies
Biotechnology Explorer™ | explorer.bio-rad.com24
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 lightCONCLUSIONS:
•Heat shock increases the transformation efficiency & competency of E. coli •Bacteria can be transformed without heat shock
65 colonies175 colonies
Biotechnology Explorer™ | explorer.bio-rad.com25
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
Biotechnology Explorer™ | explorer.bio-rad.com26
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?
Approimately95% of
plate with 50L of
AgNO3 is covered
with bacteria and cells
glow green under UV
light
Approimately95% of
plate with 50L of
AgNO3 is covered
with bacteria and cells
glow green under UV
light
Approximately 5% of plate with 500L of AgNO3 is covered with
bacteria. Isolated
colonies glow green under UV
light
Approximately 5% of plate with 500L of AgNO3 is covered with
bacteria. Isolated
colonies glow green under UV
lightApproximately 50% of plate with 100L of
AgNO3 is covered with
bacteria. Colonies glow
green under UV light
Approximately 50% of plate with 100L 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.
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.
Biotechnology Explorer™ | explorer.bio-rad.com29
Biotechnology Explorer™ | explorer.bio-rad.com30
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
Biotechnology Explorer™ | explorer.bio-rad.com31
pGlo Plasmid
•Beta Lactamase–Ampicillin
resistance
•Green Fluorescent Protein (GFP)–Aequorea
victoria jellyfish gene
•araC regulator protein–Regulates GFP
transcription
Biotechnology Explorer™ | explorer.bio-rad.com32
What is Transformation?
Uptake of foreign DNA, often a circular plasmid
E. coli cell
Biotechnology Explorer™ | explorer.bio-rad.com33
Day 1
Day 210
Transformation Procedure Overview
Biotechnology Explorer™ | explorer.bio-rad.com34
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
Biotechnology Explorer™ | explorer.bio-rad.com35
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 Procedure
Biotechnology Explorer™ | explorer.bio-rad.com36
Ca+2
heat
E. coli
Transformation
1. Transformation solution of CaCl2. Ca+2
shields negative charge of DNA phosphates
2. Incubate on iceslows fluid cell membrane
3. Heat-shockIncreases permeability of membranes
4. Nutrient broth incubationAllows beta-lactamase expression
Biotechnology Explorer™ | explorer.bio-rad.com37
Methods of Transformation
Electroporation– Electrical shock makes cell
membranes permeable to DNA
Calcium Chloride/Heat-Shock– Chemically-competent cells uptake
DNA after heat shock
Biotechnology Explorer™ | explorer.bio-rad.com38
Why perform each transformation step?
1.Transformation solution = CaCI2
Positive charge of Ca++ ions shields negative
charge of DNA phosphates
Ca++
Ca++
OCH2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
Biotechnology Explorer™ | explorer.bio-rad.com39
Why perform each transformation step?
2. Incubate on iceslows fluid cell membrane
3. Heat-shockIncreases permeability of membranes
4. Nutrient broth incubationAllows beta-lactamase expression
E. coli
Biotechnology Explorer™ | explorer.bio-rad.com40
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
Transformation Procedure Heat shock
Biotechnology Explorer™ | explorer.bio-rad.com41
Volume Measurement
Biotechnology Explorer™ | explorer.bio-rad.com42
9. Add 250ul of LB to each tube.
Leave at room temperature for 10 minutes
Transformation Procedure Recovery
Biotechnology Explorer™ | explorer.bio-rad.com43
What is Nutrient Broth?
Luria-Bertani (LB) broth
Medium that contains nutrients for bacterial growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins
Biotechnology Explorer™ | explorer.bio-rad.com44
Grow? Glow?
On which plates will colonies grow?
Which colonies will glow?
Biotechnology Explorer™ | explorer.bio-rad.com45
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!
Biotechnology Explorer™ | explorer.bio-rad.com46
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?
Biotechnology Explorer™ | explorer.bio-rad.com47
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?
Biotechnology Explorer™ | explorer.bio-rad.com48
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!
Biotechnology Explorer™ | explorer.bio-rad.com49
Green Fluorescent Protein (GFP) Chromatography Kit
GFP Purification Kit Advantages
Cloning in action
Links to biomanufacturing
Biopharmaceutical development
Amazing visual results
Biotechnology Explorer™ | explorer.bio-rad.com50
SDS PAGE Extension
Biotechnology Explorer™ | explorer.bio-rad.com51
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.comSupportWebinars
Biotechnology Explorer™ | explorer.bio-rad.com52
pGLO Plasmid
Plasmid DNA pGLO