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

3. What is Transformation?
E. coli cell
What is Transformation?
Uptake of foreign DNA from the external environment

4. A circular piece of autonomously replicating DNA in bacteria
What is a plasmid?
A circular piece of autonomously replicating DNA in bacteria
May express antibiotic resistance gene or be engineered to express genes of interest
pGLO

5. pGLO contains genes coding for 3 proteins:
Beta lactamase (bla)
Ampicillin resistance (ampR)
Green fluorescent protein (GFP)
Aequorea victoria jellyfish gene
araC repressor protein
Binds to operator & nhibits ara operon transcription

6. BOOK RETURNS TOMORROW!
Prokaryotic Gene Regulation Prokaryotic genes often are bundled into operons. The ara operon contains 3 genes (B,A,D) that code for 3 enzymes needed to break down the sugar arabinose; if arabinose is not present, the ara3 repressor binds to the operator (O) and prevents expression of the genes

7. Transformation When present, arabinose allosterically binds to the araC protein 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. Prokaryotic Gene Regulation Is the ara operon inducible or repressible?
araC
RNA Pol
Beta-lactamase

9. Transformation Regulation GFP has replaced the 3 genes in the arabinose operon, so in the presence of arabinose the GFP gene is expressed, and green fluorescent protein is produced and the bacteria GLOW!
RNA Pol
GFP

10. Transformation Procedure Overview
Day 2 (cont.)
Transformation Procedure Overview
Day 1
Day 2
Day 3 10

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

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

13. 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

14. BOOK RETURNS TOMORROW!
Prokaryotic Gene Regulation Prokaryotic genes often are bundled into operons. The ara operon contains 3 genes (B,A,D) that code for 3 enzymes needed to break down the sugar arabinose; if arabinose is not present, the ara3 repressor binds to the operator (O) and prevents expression of the genes

15. Transformation When present, arabinose allosterically binds to the araC protein 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

16. Prokaryotic Gene Regulation Is the ara operon inducible or repressible?
araC
RNA Pol
Beta-lactamase

17. Transformation Regulation GFP has replaced the 3 genes in the arabinose operon, so in the presence of arabinose the GFP gene is expressed, and green fluorescent protein is produced and the bacteria GLOW!
RNA Pol
GFP

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

19. Volume Measurement

20. 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!

21. 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?

22. 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?

23. pGLO Plasmid
pGLO
Plasmid DNA

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

25. 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