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Bacterial Transformation

Bacterial Transformation. 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). Bacterial Plasmids. Streak bacteria onto LB plates.

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Bacterial Transformation

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  1. Bacterial Transformation

  2. 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)

  3. Bacterial Plasmids

  4. Streak bacteria onto LB plates

  5. Add transformation solution CaCl2 Tubes

  6. 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

  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 Bacterial chromosomal DNA pGLO plasmids

  10. Heat-shock tubes@ 42°C for 50 seconds • Increases permeability of membrane Cell membrane Bacterial chromosomal DNA pGLO plasmids

  11. Incubate tubes on ice for 2 minutes • Returns bacteria to temperature necessary for survival Cell membrane Bacterial chromosomal DNA pGLO plasmids

  12. Add nutrient broth (LB), RT for 10 minutes • Provides nutrition for growth and gene expression GFP Bacterial chromosomal DNA 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?

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