1. pGLO Lab
Purpose: To transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar and grow in the presence of the antibiotic, ampicillin.

2. Why are bacteria a good choice for genetic transformation?
Single celled
Use plasmid DNA
Reproduce quickly

3. Gene isolated by restriction enzymes and placed into plasmid
Source of “glowing gene” for this experiment Aequorea victoria: jellyfish
Gene isolated by restriction enzymes and placed into plasmid

4. pGLO Plasmid – 3 genes Beta Lactamase Ampicillin resistance
Green Fluorescent Protein
jellyfish gene – glows
green in prescence of arabinose sugar
araC regulator protein
Makes enzyme to digest arabinose – must switch on for GFP gene to turn on!
pGLO
ori
bla
GFP
araC

5. Bacterial Transformation Plasmids enter bacterial cell and genes are expressed
Cell wall
GFP
Bacterial
chromosomal DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids

6. What is in the agar? LB – nutrient broth for bacteria to feed on
Ampicillin – antibiotic that kills bacteria
Arabinose – Sugar necessary to switch on ara C gene and the GFP gene

7. Reasons for Each
Transformation Step:
CaCl2 treatment (TS)
Positive charge of Ca 2+
neutralizes:
• negative charge of DNA
• negative charge of cell membrane

8. 2.Incubation on ice
slows cell membranes
3. Heat-shock
Increases permeability
of cell membrane
These steps allow the plasmid to be taken in by the bacteria cells

9. 4. Nutrient broth incubation
allows beta lactamase gene to be expressed (for antibiotic resistance)

10. How will we know if bacteria is transformed?
If bacteria grows in the presence of ampicillin
If bacteria glows in the presence of arabinose