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Bacterial Transformation

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  1. Bacterial Transformation BRIDGES 2014

  2. Central Dogma • DNA➔RNA➔PROTEIN➔TRAIT Phenotype Genotype

  3. Transformation • Adds new information to DNA • Cell can take up (take inside) and expresses a new piece of DNA • Transformation • New genetic information often provides organism with new trait-identifiable after transformation • Genes can be cut out of human, animal, or plant DNA and placed inside bacteria via transformation

  4. Transformation

  5. Transformation Uses • Agriculture • Frost, pest, or drought resistance • Bioremediation • Bacteria to digest oil spills • Medicine • Transforming a sick person’s cells with healthy copies of the defective gene • Creating insulin for diabetes

  6. Competent Cells • Cells that take up DNA • Cells in log phase • Growing cells take up DNA better • Transformation solution-CaCl2 • Ca2+ cationneutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane • Heat shock • Increases the permeability of the cell membrane • Duration of the heat shock is critical

  7. Plasmids • Vehicles for foreign DNA • Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival • Bacteria can transfer plasmids back and forth, allowing them to share beneficial genes • Allows for adaptation to new environments • Recent occurrence of bacterial antibiotic resistance due to transmission of plasmids

  8. Plasmids

  9. Overview • Competent cells take up DNA • DNA is brought in on plasmids • Process is called transformation

  10. Green Fluorescent Protein (GFP) • GFP gene from jellyfish- Aequoreavictoria • Causes cells to glow green under ultraviolet light • Mutations to GFP enhance fluorescence • Modified GFP gene in pGLO plasmid • After transformation bacteriaexpress the jellyfish gene and glow bright green

  11. pGLO Plasmid • Gene for GFP • Switched on by adding the sugar arabinose to the cell’s nutrient medium • Transformed cells are white on plates not containing arabinose • Transformed cells glow green when arabinose is included in the nutrient agar • Gene for resistance to the antibiotic ampicillin

  12. pGLO Plasmid • ori- origin • bla- ampicilin resistance • araC- activated by arabinose, allows down stream transcription • GFP- green fluorescent protein

  13. Activity • Add E. coli grown overnight to transformation solution • Should be in log phase • Add pGLO plasmid to +pGLO sample • Heat • Should make cells competent (take up plasmid) • Add to LB plates with ampicillin and arabinose • Grow overnight

  14. Step 1 • Label tubes • Initials • Group number • +pGLO • -pGLO

  15. Step 2 • Add 250µL transformation solution

  16. Step 3 • Put on ice

  17. Step 4 • Add E. coli grown overnight to both tubes

  18. Step 5 • Add pGLO plasmid to +pGLO tube • DO NOT ADD TO –pGLO TUBE

  19. Step 5 MODIFIED • Add pGLO plasmid to +pGLO tube • DO NOT ADD TO –pGLO TUBE • Use pipet to transfer 10µL of plasmid

  20. Step 6 • Put back on ice for 10 mins

  21. Step 7 • Label plates

  22. Step 8 • Heat Shock • Time this! • Then ice for 2 mins

  23. Step 9 • Add broth

  24. Step 9 MODIFIED • Add broth • Use broth YOU made • Using sterile technique pour some into beaker and transfer from there

  25. Step 9 MODIFIED • Add broth • Longer room temperature incubation will improve results

  26. Step 10 • Add to plates

  27. Step 11 • Spread plates • Use different spreaders for each plate

  28. Step 12 • Incubate overnight • Label with initials • At 37˚C