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pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
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DNA RNA ProteinTrait Central Framework of Molecular Biology
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Links to Real-world GFP is a visual marker Study of biological processes (example: synthesis of proteins) Localization and regulation of gene expression Cell movement Cell fate during development Formation of different organs Screenable marker to identify transgenic organisms
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Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
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Transformation: Procedure Overview Day 1 Day 2
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What is Transformation? Uptake of foreign DNA, often a circular plasmid
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What is a plasmid? A circular piece of autonomously replicating DNA Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest
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Plasmid DNA Bacterial cell Genomic DNA Bacterial DNA
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Scanning electron micrograph of supercoiled plasmid Graphic representation The Many Faces of Plasmids
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Gene Expression Beta Lactamase –Ampicillin resistance Green Fluorescent Protein (GFP) –Aequorea victoria jellyfish gene araC regulator protein –Regulates GFP transcription
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Bacterial Transformation
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Transcriptional Regulation Lactose operon Arabinose operon pGLO plasmid
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BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon RNA Polymerase ZYA ZYA LacI Effector (Lactose) ZYA LacI lac Operon Transcriptional Regulation
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RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon Gene Regulation
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Electroporation –Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock –Chemically-competent cells uptake DNA after heat shock Methods of Transformation
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Transformation Procedure Suspend bacterial colonies in Transformation solution Add pGLO plasmid DNA Place tubes on ice Heat-shock at 42°C and place on ice Incubate with nutrient broth Streak plates http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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Reasons for Performing Each Transformation Step? 1.Transformation solution = CaCI 2 Positive charge of Ca ++ ions shields negative charge of DNA phosphates
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Why Perform Each Transformation Step? 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression
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What is Nutrient Broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression –Carbohydrates –Amino acids –Nucleotides –Salts –Vitamins
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Grow? Glow? Follow protocol On which plates will colonies grow ? Which colonies will glow ?
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Results http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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Volume Measurement
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When transferring bacteria to or from an agar plate Organisms are transferred by using a sterile loop and reaching in from the side while keeping the plate covered as much as possible. This technique minimizes the risk of contamination from above. http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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When adding DNA to the transformation reaction Immerse a sterile loop into the bottle containing plasmid DNA. When the center of the loop is coated with a soap-like film, transfer it the + DNA microtube http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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