1. pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)

2. Why Teach Bacterial Transformation
Real-world connections
Link to careers and industry
Standards based
Integration with AP Big Idea 3: Molecular Genetics
Options for student inquiry based activities

3. Technology
Engineering
Math
Science
Inquiry

4. Central Framework of Molecular Biology
DNA
RNA
Protein
Trait

5. Links to Real-world GFP is a visual marker
Study of biological processes (example: synthesis of proteins)
Localization and regulation of gene expression
Cell movement
Cell fate during development
Formation of different organs
Screenable marker to identify transgenic organisms

6. Using GFP as a biological tracer
With permission from Marc Zimmer

8. 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

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

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

11. Transformation Procedure Overview
Day 1 10
Day 2

12. 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

13. 1. Label one microfuge
tube ‘+pGLO’ and
the other one ‘-pGLO’
2. Add 250 ul of Transformation solution to each tube
3. Place both tubes on ice

14. Adding bacteria and plasmid
4. Remove colony from the starter plate and add to the ‘+’ tube. Mix thoroughly.
Repeat for the ‘-’ tube.
5. Add 10ul (or one loopful) of plasmid to the ‘+’ tube only
6. Place on ice for 10 minutes

15. Transformation E. coli Ca+2
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

16. Transformation Regulation Without Arabinose
AraC
RNA Pol
Beta-lactamase

17. Transformation Regulation Arabinose operonX
RNA Pol

18. Transformation Regulation With Arabinose – induction of GFP
RNA Pol

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

20. 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

21. 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

22. 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

23. Volume Measurement

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

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

26. Preparation for plating
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!

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

28. 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!

29. Questions to consider:
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!

31. More Advanced Questions
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?

32. 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?

33. pGLO Plasmid
pGLO
Plasmid DNA

34. Extensions: Green Fluorescent Protein (GFP) Chromatography Kit 1660005EDU
GFP Purification Kit Advantages
Links to biomanufacturing
Biopharmaceutical development
Amazing visual results

35. Extensions: pGLO Kit SDS-PAGE extension 1660013EDU
pGLO SDS-PAGE Kit advantages
Learn Protein Electrophoresis
Visualize GFP on SDS-PAGE gel, it glows still!
Extensions: pGLO Kit SDS-PAGE extension EDU