Bring Inquiry into Your Classroom The 20 Question Approach

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


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)


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 . . . . ?

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


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)


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


pGLO Results


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


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


Inquiry – leading questions

What are some good “starters” for investigation questions?


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

More

Time

Student knowledge

Materials

Equipment


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

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


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


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?

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.



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?

Uptake of foreign DNA, often a circular plasmid

E. coli cell


Day 1

Day 210

Transformation Procedure Overview



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


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!



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


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?

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++


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


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


Volume Measurement


9. Add 250ul of LB to each tube.

Leave at room temperature for 10 minutes

Transformation Procedure Recovery


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?


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!


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?


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

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!


Green Fluorescent Protein (GFP) Chromatography Kit

GFP Purification Kit Advantages

Cloning in action

Links to biomanufacturing

Biopharmaceutical development

Amazing visual results


SDS PAGE Extension


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pGLO Plasmid

Plasmid DNA pGLO

Documents

Bring Inquiry into Your Classroom The 20 Question Approach