BACTERIAL TRANSFORMATION TRAINING F LUORESCENT P ROTEIN A CTIVITIES Bacterial Transformation Protein Purification Discovery of GFP-1960s Aequorea victoria OSAMU SHIMOMURA Co-winner of Nobel Prize How Fluorescence Works Bioluminescent organism produces its own light. A fluorescent organism absorbs light at one wavelength (UV) and a re- emits the light at a visible wavelength= color Scorpion- UV Light Scorpion- Natural LightNatural Light In the Dark BioluminescenceFluorescence Bioluminescence vs. Fluorescence Many organisms have the ability to fluoresce Many organisms have the ability to fluoresce Jellyfish Amphipod Spiders palps Roger Tsien and Rainbow Proteins E. COLI What is Transformation? Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome What is a plasmid? A small circular piece of DNA Naturally occurring Can be altered in lab to express protein of interest What is Transformation? Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome What is Transformation? Bacterial chromosome Allow bacteria to grow for 1-3 days on plate with ampicillin. Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome What is Transformation? Bacteria now express cloned fluorescent protein Bacterial chromosome Allow bacteria to grow for 1-3 days on plate with ampicillin. Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome How are plasmids engineered? Host DNA fragments (i.e. coral or jellyfish FP coding DNA) DNA Plasmid Vector Cut plasmids open with restriction enzymes Cut genomic DNA into fragments + Ligate (paste) fragments into cut DNA vector Screen for and select plasmid containing FP gene Bacterial Transformation Procedure Shielding the charge Ca ++ O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++ CaCl 2 Positive charge of Ca ++ ions shields negative charge of DNA phosphates Stress through heat Incubate on ice slows fluid cell membrane Heat-shock increases permeability of membranes Leave in heat 45 seconds!!! Too short, and bacteria won't let in plasmid. Too long, and the bacteria will die. Why Ampicillin? Ampicillin inhibits cell growth. Only cells that can inactivate the ampicillin around them will grow. Ampicillin resistance is tied to (expressed with) the fluorescent protein gene Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate MITOSIS a similar process in diverse species ...using various organisms to understand humans: Frog Egg Extract + sperm DNA A. Desai Frog Cell C.E. Walczak Marsupial Cell S.L. Kline Fly Embryo T. Megraw Frog Egg Extract + DNA-coated beads R. Heald Human Cell J. Waters Worm Embryo I.M. Cheeseman Fluorescent Proteins-Applications The rainbow of mFruit Fluorescent Proteins