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

3. Lab Timeline Introduction /Transformation
Transform bacteria with pGLO plasmid
When a cell takes up and expresses a new piece of trait:
Uses: plants to be resistent to pest
Bacterial to digest oil spills,
Gene therapy

4. Central Framework of Molecular Biology: The Central Dogma
DNA
RNA
Protein
Trait

5. Context Genetic engineering of organism Use of experimental controls
Interpretation of experimental results
Calculate transformation efficiency
GMO
Cell biology
Evolution: antibiotic resistance, selection mechanism, adaptation
Genetics: Central Dogma, gene regulation, lac operon

6. GRP (green fluorescent protein from jelly fish!)

7. 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

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

9. What is Transformation?
GFP
Uptake of foreign DNA, often a circular plasmid
Beta-lactamase
Ampicillin
Resistance

10. 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

11. Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA

12. The Many Faces of Plasmids
Graphic representation
Scanning electron micrograph of supercoiled plasmid

13. Gene Expression Beta Lactamase Ampicillin resistance
Green Fluorescent Protein (GFP)
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription

14. Bacterial Transformation
Cell wall
GFP
Bacterial
chromosomal DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids

15. Transcriptional Regulation
Lactose operon
Arabinose operon
pGLO plasmid

16. Transcriptional Regulation
RNA Polymerase Z Y A
LacI
Effector (Lactose)
lac Operon B A D
araC
RNA Polymerase
Effector (Arabinose)
ara Operon

17. Gene Regulation B A D ara Operon ara GFP Operon GFP Gene
araC
RNA Polymerase
Effector (Arabinose)
ara Operon
RNA Polymerase
araC
ara GFP Operon
GFP Gene
Effector (Arabinose)

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

19. Transformation Procedure Overview
Day 1
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
Day 2

20. Reasons for Performing 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?
Cell wall
GFP
2. Incubate on ice
slows fluid cell membrane
3. Heat-shock
Increases permeability of membranes
4. Nutrient broth incubation
Allows beta-lactamase
expression
Beta-lactamase
(ampicillin resistance)

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

23. Grow? Glow? Follow protocol On which plates will colonies grow?
LB/Amp
Follow protocol
On which plates will colonies grow?
Which colonies will glow?
LB/Amp/Ara LB

24. Lab Safety The E. coli is not pathogenic Safety Procedure:
Decontaminate work surface each day
All liquid or solid waste needs to be decontaminated (using bleach!)
Wash hands after handling bacteria and before leaving lab
Carefully following all protocol / procedure.
If you are allergic to ampicillin…let me know!
UV lamp do not stare into the light or shine on your own skin!

25. Volume Measurement

26. Lab technique Sterile technique!
Do not introduce contaminating bacteria
The inoculation loops, pipets, agar plates should NOT touch or be placed onto contaminating surfaces!
Wash your Hands!!
Be careful with timing…it’s the most important part of the lab!!