1. Modeling Transformation What does each step do?

2. Transformation Procedure

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

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

6. E. coli Cell Bacterial chromosomal DNA Cell membrane - - - - - - - - - - - - - - Ribosomes

7. Add transformation solution Tube CaCl 2

8. Transformation solution: CaCl 2 Positive charge of Ca ++ ions shields negative charge of DNA Positive charge of Ca ++ ions shields negative charge of DNA O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++

9. Inoculate with bacteria Tube CaCl 2

10. Add plasmid Tube CaCl 2

11. Incubate on ice Slows movement of cell membrane Slows movement of cell membrane pGLO plasmids Bacterial chromosomal DNA Cell membrane

12. Heat-shock @ 42°C Increases permeability of membrane Increases permeability of membrane pGLO plasmids Bacterial chromosomal DNA Cell membrane

13. Incubate on ice Returns bacteria to temperature necessary for survival Returns bacteria to temperature necessary for survival pGLO plasmids Bacterial chromosomal DNA Cell membrane

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

15. What do you expect to happen?

19. - pGLO +pGLO LBLB/amp LB/amp/a rabinose