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

3. 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

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

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

6. Transformation Regulation Without arabinose the GFP gene is NOT transcribed, so no GFP is produced (this is an example of an INDUCIBLE operon in which arabinose is the inducer)
araC
RNA Pol
Beta-lactamase

7. Transformation Arabinose interacts with the araC protein (a repressor) to enable RNA polymerase to bind to the promoter and transcribe the 3 genes in the arabinose operon, producing the enzymes necessary to digest arabinose
Arabinose
araC X
RNA Pol

8. Transformation Regulation GFP has replaced the 3 genes in the arabinose operon, so in the presence of arabinose the GFP gene is expressed, and GFP is produced (translation: the bacteria GLOW under UV light!)
RNA Pol
GFP

9. Transformation Procedure Overview
Day 2
Transformation Procedure Overview
Day 1
Day 3 10

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

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

12. Transformation E. coli Ca+2
Transformation solution of CaCl2. Ca2+ 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

13. Transformation Regulation Without arabinose the GFP gene is NOT transcribed, so no GFP is produced (this is an example of an INDUCIBLE operon in which arabinose is the inducer)
araC
RNA Pol
Beta-lactamase

14. Transformation Arabinose interacts with the araC protein (a repressor) to enable RNA polymerase to bind to the promoter and transcribe the 3 genes in the arabinose operon, producing the enzymes necessary to digest arabinose
Arabinose
araC X
RNA Pol

15. Transformation Regulation GFP has replaced the 3 genes in the arabinose operon, so in the presence of arabinose the GFP gene is expressed, and GFP is produced (translation: the bacteria GLOW under UV light!)
RNA Pol
GFP

16. On which plate(s) will colonies grow?
On which plate(s) will colonies GLOW?
LB = nutrient broth
amp = ampicillin
ara = arabinose

17. Volume Measurement

18. 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!

19. 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?

20. 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?

21. pGLO Plasmid
pGLO
Plasmid DNA

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

23. 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