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Modeling Transformation

Modeling Transformation. What does each step do?. Transformation Procedure. Transformation Procedure. Building Your Model. Yarn = chromosomal DNA Beads = ribosomes Black velcro = plasmid DNA Green velcro = GFP gene Pink velcro = Ampicillin resistance gene Bottle = cell membrane

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Modeling Transformation

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  1. Modeling Transformation What does each step do?

  2. Transformation Procedure

  3. Transformation Procedure

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

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

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

  7. Add transformation solution CaCl2 Tube

  8. Transformation solution: CaCl2 Ca++ O Ca++ • Positive charge of Ca++ ions shields negative charge of DNA O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH

  9. Inoculate with bacteria CaCl2 Tube

  10. Add plasmid CaCl2 Tube

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

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

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

  14. Add nutrient broth (LB) • Provides nutrition for growth and gene expression GFP Bacterial chromosomal DNA 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?

  19. What do you expect to happen? -pGLO +pGLO LB/amp LB LB/amp LB/amp/arabinose

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