300 likes | 429 Vues
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?
E N D
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? • 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
What are we going to do monday? • Insert the foreign DNA into bacteria (transformation) to make them glow
Discovery of GFP-1960’s Aequorea victoria OSAMU SHIMOMURA Co-winner of Nobel Prize
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
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.
Many organisms have the ability to fluoresce www.worldnetcams.com/sealife/cerianthus.jpg Jellyfish Amphipod Spider’s palps
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
Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid by bacteria Plasmid
What is a plasmid? • A small circular piece of DNA • Naturally occurring • Can be altered in lab to express protein of interest
Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid
Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid
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
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…
Central Dogma Genotype DNA transcription mRNA translation Protein Phenotype Trait
Bacterial Transformation Procedure • Add ingredients: -bacteria -plasmid -CaCl 2. Cool & then heat shock
CaCl2- Shields the negative charges • Positive charge of Ca++ ions shields negative charge of DNA and phospholipid heads
Temperature Changes • Incubate on ice slows fluid cell membrane, so the Ca+ can shield negative charges • Heat-shock increases permeability of membranes
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.
Why Ampicillin? • Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate
Why do we useFluorescent Proteins? • To view parts of organisms or cells, to learn more about them
Fluorescent Proteins-Applications ……...using various organisms to understand humans:
House Mouse (Mus musculus) Transgenic Organisms Under Black Light • Scientists can also cause organisms to fluoresce! Zebra Fish (Danio rerio) Under normal light
Transformation Applications • To make medicines -e.g.insulin • To make other commercially proteins - e.g. proteins that decompose oil
Transformation video • BioBridge Transformation Video
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.