2. Transformation Procedure Overview
Day 1
Day 2

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

4. Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA

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

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

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

8. Transcriptional Regulation
Lactose operon
Arabinose operon
pGLO plasmid

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

10. 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)

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 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. 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

14. 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)

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

16. 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