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Friday, April 11

Friday, April 11. AGENDA: Bacterial Transformation Lecture Reading Time. Important Vocabulary. What is transformation? The process by which the genetic makeup of an organism is altered by the insertion of a new gene (or exogenous DNA) into its genome.  What is a plasmid?

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Friday, April 11

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  1. Friday, April 11 • AGENDA: • Bacterial Transformation Lecture • Reading Time

  2. Important Vocabulary • What is transformation? • The process by which the genetic makeup of an organism is altered by the insertion of a new gene (or exogenous DNA) into its genome.  • What is a plasmid? • A circular piece of DNA that is separate from and replicates independently of the chromomsal DNA • “DNA donuts” • What is a recombinant plasmid? • A plasmid that has a section of foreign DNA added to it • What is an antibiotic? • A substance that can inhibit the growth of or kill bacteria

  3. What are we going to do monday? • Insert the foreign DNA into bacteria (transformation) to make them glow

  4. Discovery of GFP-1960’s Aequorea victoria OSAMU SHIMOMURA Co-winner of Nobel Prize

  5. RFP Isolated from Propionibacteriumfreudenreichii in 1998

  6. How Fluorescence Works Fluorescence- absorbing light at one wavelength (UV) and a re-emits the light at a visible wavelength= color www.worldnetcams.com/sealife/cerianthus.jpg

  7. Scorpion- Natural Light Scorpion- UV Light Bioluminescence vs. Fluorescence Bioluminescence Fluorescence http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/ Natural Light In the Dark A fluorescent organism absorbs light at one wavelength (UV) and a re-emits the light at a visible wavelength= color Bioluminescent organism produces its own light.

  8. Many organisms have the ability to fluoresce www.worldnetcams.com/sealife/cerianthus.jpg Jellyfish Amphipod Spider’s palps

  9. Roger Tsien and Rainbow Proteins

  10. E. coli

  11. Model Organisms C. elegans - genetically mapped and able to target specific cells for study Drosophila– most common model organism Lab mouse – easy to alter genetically • Model organisms: Used in many scientific experiments • Easy to manipulate for experiments • Short life-cycle • Easy to keep alive • Short generation times E. coli – easy to transform

  12. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid by bacteria Plasmid

  13. What is a plasmid? • A small circular piece of DNA • Naturally occurring • Can be altered in lab to express protein of interest

  14. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

  15. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

  16. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days on plate with ampicillin. Bacterial chromosome

  17. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days on plate with ampicillin. Bacterial chromosome Bacteria now express cloned fluorescent protein…

  18. Central Dogma Genotype DNA transcription mRNA translation Protein Phenotype Trait

  19. Bacterial Transformation Procedure • Add ingredients: -bacteria -plasmid -CaCl 2. Cool & then heat shock

  20. CaCl2- Shields the negative charges • Positive charge of Ca++ ions shields negative charge of DNA and phospholipid heads

  21. Temperature Changes • Incubate on ice slows fluid cell membrane, so the Ca+ can shield negative charges • Heat-shock increases permeability of membranes

  22. Antibiotic Resistance • Antibiotics: molecules that stop bacteria from reproducing. • Antibiotic resistance: the ability of a bacteria to inactivate the antibiotic. • Antibiotic resistance gene: segment of DNA that codes for a protein that inactivates an antibiotic.

  23. Why Ampicillin? • Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate

  24. Why do we useFluorescent Proteins? • To view parts of organisms or cells, to learn more about them

  25. Fluorescent Proteins-Applications ……...using various organisms to understand humans:

  26. House Mouse (Mus musculus) Transgenic Organisms Under Black Light • Scientists can also cause organisms to fluoresce! Zebra Fish (Danio rerio) Under normal light

  27. Transformation Applications • To make medicines -e.g.insulin • To make other commercially proteins - e.g. proteins that decompose oil

  28. The rainbow of mFruit Fluorescent Proteins

  29. Transformation video • BioBridge Transformation Video

  30. Lab Prep • Shoes! Cover them toes! • Hair! On Mondays, we wear ponytails. • Food! Don’t bring it in here! • Drink! Water is fine as long as it is in the “safe zone” • Read! Read your lab through first. DO IT.

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