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Genetic Transformation

Genetic Transformation. WHY SHOULD WE DO THIS?. Genetic Engineering is now widely used:. Bacteria that produce human insulin. Corn that produces insecticide. Rice that produces extra vitamin A. Goats that produce spider silk. IMPORTANT TERMS. Promoter - Plasmid - Transformation

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Genetic Transformation

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  1. Genetic Transformation

  2. WHY SHOULD WE DO THIS? Genetic Engineering is now widely used: • Bacteria that produce human insulin • Corn that produces insecticide • Rice that produces extra vitamin A • Goats that produce spider silk

  3. IMPORTANT TERMS Promoter - Plasmid - Transformation Competent- an “on/off” switch for a gene a small, circular piece of bacterial DNA that is not part of the chromosome a process in which bacteria take up DNA from their environment • can be triggered by electric shock or heat shock • able to be transformed

  4. What is transformation? • When a cell takes upand expressesa new piece of genetic material—DNA— in order to change the organism’s traits • A geneis a piece of DNA which provides the instructions for making (coding for) a particular protein Protein Trait DNA

  5. GFP Gene • found in jellyfish • engineered into bacteria Green Fluorescent Protein GLOWING CELLS Protein Trait DNA

  6. History of Transformation • 1928 – Fredrick Griffth first described transformation • Pneumococcus bacteria (pneumonia) and mice • 1944 – Oswald Avery, Colin MacLeod, Maclyn McCarty identified the “transforming principle” as DNA

  7. History of Transformation • 1952 – Alfred Hershey and Martha Chase provided final proof that DNA was the molecule of heredity • 1970 – Morton Mandel and Akiko Higa developed a protocol for transforming E. coli bacteria

  8. What is transformation used for? • Agricultural • Genes coding for traitssuch as frost, pest or drought resistance can be genetically transformed into plants

  9. What is transformation used for? • Environmental • Bacteria can be genetically transformed with genes enabling them to digest oil spills or removepollutants from the environment

  10. What is transformation used for? • Medical • Production of human proteins to treat genetic diseases

  11. What are we doing? • We will transform bacteria (E. coli), giving it the ability to make green fluorescent proteins

  12. Green Fluorescent Protein • Used in science as a visual marker… • Biological processes (protein production) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Marker to identify transgenic organisms

  13. How does it work? Cell membrane Bacterial chromosomal DNA *plasmids*

  14. Making Plasmids = Plasmid + Gene

  15. STARTING MATERIALS E. coli cells • sensitive to antibiotics • can’t glow • competent - able to be transformed Bacterial chromosome

  16. AmpR Ara promoter STARTING MATERIALS • Plasmid containing: • Ampicillin resistance gene (always expressed) • Ara promoter - turned on in the presence of arabinose

  17. GFP gene STARTING MATERIALS Jellyfish DNA GFP = Green Fluorescent Protein glows under UV light

  18. AmpR Ara GFP STARTING MATERIALS E. coli cells Plasmid Jellyfish DNA

  19. AmpR GROW ON AN AGAR PLATE GFP Ara … that can GLOW! END RESULT Recombinant Bacteria…

  20. makes all transformed bacteria resistant to ampicillin controls GFP gene expression only turned on in the presence of arabinose HOWEVER… things are actually a bit more complex. AmpR pGLO plasmid GFP Ara promoter

  21. Steps of Transformation Step 1 DNA Isolation Isolation of Your Gene of Interest (GFP) Step 2 Recombinant DNA Insertion of foreign DNA into bacterial plasmid using restriction enzymes and DNA ligase Step 3 Transformation Insertion of recombinant DNA into bacteria by making bacteria competent Use CaCl2 and heat shock techniques

  22. Transformation Process • 1. Place cells in transformation solution (CaCl2 – Calcium chloride) • the positive ions in calcium chloride neutralizes the negative charges of the DNA backbone and the phospholipids and allows DNA to enter the cells

  23. Transformation Process • 2. Heat shock cells • The increased heat increases the permeability of the membrane, allowing the DNA to enter more easily. (Makes pores bigger) • Also, a temperature imbalance is created between the inside/outside of cell, causing DNA to enter cell. • 3. Ice bath • The sudden drop in temperature “freezes” the pores open allowing DNA to enter more easily.

  24. pGLO Plasmid To make green fluorescent proteins GFP Promoter araC amp To survive with Ampicillin(antibiotic)

  25. Promoters • Promoter: a sequence that “turns on” a gene • Arabinose: a simple sugar • araC promoter: turns on the GFP gene in the presence of arabinose sugar • if arabinose sugar is ABSENT, the GFP gene DOES NOT get turned on • when arabinose sugar is PRESENT, the GFP gene turns ON.

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