1. Bacterial Transformation

2. What is transformation?
Uptake of foreign DNA, often a circular plasmid
GFP
Amp Resistance

3. What is a plasmid? A circular piece of autonomously replicating DNA
ori
bla
Originally evolved by bacteria
May express antibiotic resistance gene or
be modified to express proteins of interest

4. pGlo plasmid Beta Lactamase Green Fluorescent Protein
Ampicillin resistance The beta lactamase protein is produced and secreted by bacteria that contain the plasmid. Inactivates ampicillin.
Green Fluorescent Protein
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription
pGLO
ori
bla
GFP
araC

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

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

7. Transformation procedure
Suspend bacterial colonies in Transformation Solution
Add pGLO plasmid DNA
Place tubes on ice
Heat shock at 42oC and place on ice
Incubate with nutrient broth
Streak plates

8. Reasons for performing each transformation step
Ca++ O
CH2 P
Base OH
Sugar
Transformation solution = CaCl2
pH = 6.1
Positive charge of Ca+2 ions neutralizes negative charge of phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to enter the cells.

9. Why perform each transformation step?
Incubation on ice slows fluid cell membranes
Heat-shock increases the permeability of cell membrane to DNA. Increase the bacterial uptake of foreign DNA.
Nutrient broth incubation allows beta lactamase expression
GFP
Cell wall
Beta lactamase
(ampicillin resistance)

10. What is nutrient broth or LB broth?
Luria-Bertani (LB) broth
Medium that contains nutrients for bacterial growth and gene expression
carbohydrates
amino acids
nucleotides
salts
vitamins

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

12. Central Framework of Molecular Biology
DNA
RNA
Protein
Trait