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Understanding Bacterial Transformation and Fluorescent Proteins

In this session, we will explore the concept of bacterial transformation, where foreign DNA is introduced into bacteria to alter their genetic makeup. Key topics include the role of plasmids, the definition and implications of antibiotics, and how transformed bacteria can be used as models for scientific experiments. We will also discuss the discovery of fluorescent proteins, their applications in biology, and conduct a hands-on transformation experiment involving plasmids. This interactive learning experience will illuminate the processes behind genetic engineering and fluorescence.

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Understanding Bacterial Transformation and Fluorescent Proteins

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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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