BACTERIAL TRANSFORMATION Laboratory Introduction What is a protocol?What is a model organism? Why do scientists use protocols? What are some examples of model organisms? Now lets practice pipetting! Drosophila most common model organism E. coli easy to transform Easy to manipulate for experiments Easy to keep alive Short life-cycle Short generation times C. elegans genetically mapped; can target specific cells for study Lab mouse easy to alter genetically Model Organisms Escherichia coli Green Fluorescent Protein (GFP) Osamu Shimomura Co-winner of Nobel Prize Aequorea victoria Discovery of GFP (1960s) How does fluorescence work? Green light (Lower energy) Excited state Ground state Blue light (High energy) Fluorescence vs. Bioluminescence Scorpion- UV Light Scorpion- Natural Light Fluorescent organism: Absorbs light at one wave- length (UV) and re-emits light at a visible wavelength (color) Bioluminescent organism: Produces its own light. Natural Light In the Dark Fluorescence vs. Bioluminescence Fluorescent organism: Absorbs light at one wave- length (UV) and re-emits light at a visible wavelength (color) Bioluminescent organism: Produces its own light. Jellyfish Amphipod Spiders palps Fluorescent Organisms Corals GFP RFP Roger Tsien and Rainbow Proteins MITOSIS a similar process in diverse species 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 Protein Applications E. coli bacterial cell Bacterial genome Plasmid Uptake of foreign DNA, often a circular plasmid What is Transformation? A small circular piece of DNA Naturally occurring Can be altered in lab to express protein of interest What is a plasmid? Host DNA fragments (i.e. coral or jellyfish FP coding DNA) DNA Plasmid Vector Cut plasmid open with restriction enzymes Cut genomic DNA into fragments + Ligate (paste) fragments into cut DNA vector Screen for and select plasmid containing FP gene How are plasmids engineered? E. coli bacterial cell Bacterial genome Plasmid Cell produces protein coded by plasmid DNA. What is Transformation? DNARNAProtein Bacterial Transformation Procedure Step 1: Collect bacterial colonies Like scraping whipped cream off of Jello. Ca ++ O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++ Step 2: Put bacteria in CaCl 2 Positive charge of Ca 2+ ions shields negative charge of DNA phosphates Step 3: Add Plasmid to (+) Tube Amp R Ampicillin resistance gene FP gene Plasmid Mix 1 GFP BFP Grape Plasmid Mix 2 YFP Tangerine Cherry Incubate on ice to slow fluid cell membrane Heat-shock increases permeability of membranes Step 4: Heat Shock! Leave in heat 45 seconds!!! - Too short, and bacteria won't let in plasmid. - Too long, and the bacteria will die. ICE HEAT ICE Step 5: Plate on Ampicillin cell wallAmpicillin inhibits cell wall 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. We have two controls LB only (-) = Control 1: check for viable cells LB amp (-) = Control 2: check for ampicillin function LB amp (+) = Experimental plate: grow transformants We have six colors Plasmid Mix 1Plasmid Mix 2 GFP BFP GrapeYFP Tangerine Cherry Bacterial Transformation Procedure