1. Modeling Transformation
What does each step do?

2. Transformation Procedure
Suspend bacterial colonies in Transformation solution (CaCl)
Add pGLO plasmid DNA
Place tubes on ice
Heat-shock at 42°C and place on ice
Incubate with nutrient broth
Streak plates

3. Transformation Procedure
Suspend bacterial colonies in Transformation solution (CaCl)
Add pGLO plasmid DNA
Place tubes on ice
Heat-shock at 42°C and place on ice
Incubate with nutrient broth
Streak plates

4. Building Your Model Yarn = chromosomal DNA Sequins = ribosomes
Black velcro = plasmid DNA
Green velcro = GFP gene
Pink velcro = beta-lactamase gene
Bottle = cell membrane
Pipe cleaner = flagellum
Cup = micro-tube

5. 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)

6. E. coli Cell - Cell membrane Bacterial chromosomal DNA Ribosomes
Place negative signs on outside of bottle
Put yarn and sequins inside bottle, close cap.

7. Add transformation solution
CaCl2
Tube

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

9. Inoculate with bacteria
CaCl2
Tube

10. Add plasmid
CaCl2
Tube

11. Incubate on ice Slows fluid cell membrane Cell membrane Bacterial
pGLO plasmids
Bacterial
chromosomal DNA
Cell membrane
Remove cap

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

13. Incubate on ice 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

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

15. What do you expect to happen?

16. What do you expect to happen?

17. What do you expect to happen?

18. What do you expect to happen?