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pGLOTM Bacterial Transformation Courtesy BioRad Corporation
4/21/2017
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pGLO/ Green Fluorescent Protein Isolation A single bacteria might be worth 5 billion dollars to the right buyer.
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Central Framework of Molecular Biology
DNA RNA Protein Trait 4/21/2017
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What is transformation?
Uptake of foreign DNA, often a circular plasmid, from the surroundings with the expression of these genes. GFP Amp Resistance 4/21/2017
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What is a plasmid? A circular piece of autonomously replicating DNA
ori bla Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest 4/21/2017
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Uses of Genetic Transformation
Agriculture Introduction of drought, pest, or frost resistance genes “BT Corn” Bioremediation Introduction of genes into bacteria giving them the ability to degrade hydrocarbons, “Oil Eating Bacteria” Medicine Gene therapy Production of human proteins Insulin, clotting factors 4/21/2017
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pGLO Plasmid-Draw this!!
Beta Lactamase Ampicillin resistance Green Fluorescent Protein Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription pGLO ori bla GFP araC 4/21/2017
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Purpose of pGLO Transformation
To transform E. coli K-12 with a plasmid that contains a gene that codes for Green Fluorescent Pigment (GFP) Transformed cells will produce GFP and glow a brilliant green under UV light. 4/21/2017
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Work of the pGLO Plasmid
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Arabinose Operon RNA Polymerase Regulatory Gene--araC Gene products
Binds RNA polymerase Gene products 4/21/2017 RNA Polymerase
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Transcriptional Regulation
RNA Polymerase araC ara GFP Operon GFP Gene ara Operon araC B A D Effector (Arabinose) Effector (Arabinose) araC B A D RNA Polymerase araC B A D 4/21/2017
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Transformation Procedure
Suspend bacterial colonies in Transformation Solution Add pGLO plasmid DNA Place tubes on ice Heat shock at 42oC and place on ice Incubate with nutrient broth Streak plates 4/21/2017
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Transformation Procedures Day 1 Day 2 4/21/2017
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Reasons for performing each transformation step?
Ca++ O CH2 P Base OH Sugar Transformation solution = CaCl2 Positive charge of Ca+2 ions shields negative charge of DNA phosphates 4/21/2017
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Why perform each transformation step?
Incubation on ice slows fluid cell membranes Heat-shock increases permeability of cell membrane Incubation in LB nutrient broth allows beta lactamase expression GFP Cell wall Beta lactamase (ampicillin resistance) 4/21/2017
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Inducible, Repressible or Both??
4/21/2017
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The Gel Analysis 4/21/2017
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Graphing Migration Distance
4/21/2017
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