Transcript Bring Inquiry into Your Classroom with the pGLO Plasmid - Bio-Rad

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
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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)
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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?
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pGLO
 What do we already know?
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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)
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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.
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Transformation Procedure
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What is the point of . . . . ?
1. Calcium Chloride in
transformation solution: Ca+2
shields negative charge of
DNA phosphates
2. Ice incubation: slows fluid
cell membrane
Ca+2
heat
3. Heat-shock: increases
permeability of membranes
4. Nutrient broth incubation:
Allows beta-lactamase
expression
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Regulation of GFP – no arabinose
Without
arabinose:
AraC
• AraC is inactive
RNA
Pol
• RNA pol
doesn’t bind
and transcribe
GFP
• (bla and araC
are still
transcribed)
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Betalactamase
Regulation of GFP – with arabinose
With
arabinose:
• AraC is active
and helps RNA
pol bind
• RNA pol
transcribes
GFP
• GFP is
produced –
bacteria glow
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RNA Pol
pGLO Results
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pGLO
 What kinds of questions can we ask?
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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?
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pGLO
 More?
Question
Expert? Big idea?
Investigation?
Which plate should have growth / glow? What happens
if we don’t use LB/Amp plates?
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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
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Inquiry – leading questions
 What are some good “starters” for
investigation questions?
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Student Plan for Inquiry Investigation
Question
Research
Independent, Dependent, Controlled variables
Hypothesis
Materials / equipment needed
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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?
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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?
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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.
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Less
Time
Student
knowledge
Materials
Equipment
More
pGLO Inquiry – Level 1
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Higher Level Inquiry
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Inquiry in Action
Amy Inselberger and Shari Cohen with student volunteers
from Stevenson HS
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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 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
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Bacterial
65 colonies
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
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
175 colonies
65 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
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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
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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
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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?
Approimately
95% of plate
with 50mL of
AgNO3 is
covered with
bacteria and
cells glow
green under
UV light
Approximately 5%
of plate with
500mL of AgNO3 is
covered with
bacteria. Isolated
colonies 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.
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 A circular piece
of autonomously
replicating DNA
What is a plasmid?
 Originally
evolved by
bacteria
 May express
antibiotic
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•Beta
Lactamase
–Ampicillin
resistance
pGlo Plasmid
•Green
Fluorescent
Protein (GFP)
–Aequorea
victoria jellyfish
gene
•araC
regulator
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protein
E. coli cell
 Uptake of foreign
DNA, often a
circular plasmid
What is
Transformation?
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Transformation
Procedure Overview
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Day 1
Day 2
 Suspend bacterial colonies in
transformation solution
 Add pGLO plasmid DNA
Transformation
Procedure
 Place tubes on ice
 Heat-shock at 42°C and place on ice
 Incubate with nutrient broth
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 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!
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E. coli
1. Transformatio
n solution of
CaCl2. Ca+2
shields negative
charge of DNA
phosphates
Transformation
2. Incubate on
ice
slows fluid cell
membrane
3.
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Heatshock
Increases
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permeability of
Ca+2
heat
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 Electroporation
– Electrical shock makes cell
membranes permeable to DNA
Methods of
Transformation
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 Calcium Chloride/Heat-Shock
– Chemically-competent cells uptake
DNA after heat shock
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Ca++
Ca++
O
O P O
O
CH2
Base
O
Sugar
Why perform
1.Transformation
each
transformation
solution = CaCI2
step?
Positive charge of
Ca++ ions shields
negative
charge of DNA
phosphates
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O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
E. coli
Incubate on
Why ice
perform
slows fluid cell
eachmembrane
transformation
step?
3. Heat-shock
Increases
permeability of
membranes
2.
4. Nutrient
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broth
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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
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Volume
Measurement
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Transformation Procedure
Recovery
9. Add 250ul of LB to each tube.
Leave at room temperature for 10
minutes
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 Luria-Bertani (LB) broth
 Medium that contains nutrients for bacterial growth
and gene expression
– Carbohydrates
Amino acids
What is– Nutrient
Broth? – Nucleotides
– Salts
– Vitamins
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Grow?
Glow?
 On which plates will colonies grow?
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Questions to consider:
 How important is each step in the lab
protocol?
Student Inquiry
 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?
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– Importance of controlling other variables
– Collaborative approach / share data
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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?
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 Can I remove the regulation of GFP so
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 What materials and equipment do I
have on hand, and what will I need to
order?
Student
Inquiry
Teacher
Considerations
– 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?
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– Limited time? Have students read lab and
explorer.bio-rad.comcome up with inquiry questions and protocol
before they start. Collaborative approach.
Transformation Procedure
Plating Bacteria
New
pipet!
10. Put 100 ul of solution
onto the appropriate
plates
New
loop!
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11. Streak plates
GFP Purification Kit Advantages
 Cloning in action
 Links to biomanufacturing
Green Fluorescent
Protein (GFP)
Chromatography Kit
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 Biopharmaceutical development
 Amazing visual results
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SDS PAGE Extension
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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
51
 From singleplex to multiplex: making
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Plasmid DNA
pGLO Plasmid
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pGLO