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BACTERIAL TRANSFORMATION TRAINING
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F LUORESCENT P ROTEIN A CTIVITIES Bacterial Transformation Protein Purification
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Discovery of GFP-1960’s Aequorea victoria OSAMU SHIMOMURA Co-winner of Nobel Prize
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www.worldnetcams.com/sealife/cerianthus.jpg How Fluorescence Works
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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 Light http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/ Natural Light In the Dark BioluminescenceFluorescence Bioluminescence vs. Fluorescence
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www.worldnetcams.com/sealife/cerianthus.jpg Many organisms have the ability to fluoresce Many organisms have the ability to fluoresce Jellyfish Amphipod Spider’s palps
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Roger Tsien and Rainbow Proteins
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E. COLI
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What is Transformation? Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome
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What is a plasmid? A small circular piece of DNA Naturally occurring Can be altered in lab to express protein of interest
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What is Transformation? Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome
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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
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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
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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
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Bacterial Transformation Procedure
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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
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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.
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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
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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
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The rainbow of mFruit Fluorescent Proteins
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