1. Bacterial Transformation

2. Real image of a bacterial plasmid (Scanning electron micrograph)
pGLO Plasmid
GFP
araC
GFP: gene for Green Fluorescent Protein
araC: gene for Beta- lactamase (Ampicillin resistance)
Make plasmid
Real image of a bacterial plasmid (Scanning electron micrograph)

3. Bacterial Plasmids

4. Streak bacteria onto LB plates

5. Add transformation solution
CaCl2
Tubes

6. Transformation solution: CaCl2
Positive charge of Ca++ ions shields negative charge of DNA O
CH2 P
Base OH
Sugar
Put bacterium, plasmid, and in tube, remove negative signs from bottle.
Positive charge of Ca++ ions shields negative charge of DNA phosphates

7. Inoculate 2 tubes with bacteria
CaCl2
Tubes

8. Add pGLO plasmid to one tube only
CaCl2
Tube

9. Incubate tubes on ice Slows movement of cell membrane Cell membrane
pGLO plasmids
Bacterial
chromosomal DNA
Cell membrane
Remove cap

10. Heat-shock tubes@ 42°C for 50 seconds
Increases permeability of membrane
Bacterial
chromosomal DNA
Cell membrane
Put tube in the “hot water” bath and push the plasmid into the cell
Increases permeability of membrane
pGLO plasmids

11. Incubate tubes on ice for 2 minutes
Returns bacteria to temperature necessary for survival
pGLO plasmids
Bacterial
chromosomal DNA
Cell membrane
Put tube in the “hot water” bath and push the plasmid into the cell

12. Add nutrient broth (LB), RT for 10 minutes
Provides nutrition for growth and gene expression
Bacterial
chromosomal DNA
GFP
Beta lactamase
(ampicillin resistance)
pGLO plasmids

13. Streak Plates with bacteria

14. What is an operon?

15. Arabinose operon

16. Arabinose operon and GFP

17. What do you expect to happen?