TM pGLO Bacterial Transformation Courtesy BioRad Corporation 1 3/14/2016 pGLO/ Green Fluorescent Protein Isolation A single bacteria might be worth 5 billion dollars to the right buyer. Central Framework of Molecular Biology DNA 3 RNA Protein Trait 3/14/2016 What is transformation? Uptake of foreign DNA, often a circular plasmid, from the surroundings with the expression of these genes. GFP Amp Resistance 4 3/14/2016 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 5 3/14/2016 Uses of Genetic Transformation Agriculture Bioremediation Gene therapy Production of human proteins 6 Introduction of genes into bacteria giving them the ability to degrade hydrocarbons, “Oil Eating Bacteria” Medicine Introduction of drought, pest, or frost resistance genes “BT Corn” Insulin, clotting factors 3/14/2016 pGLO PlasmidDraw this!! Beta Lactamase • Ampicillin resistance Green Fluorescent Protein • Aequorea victoria jellyfish gene araC regulator protein • Regulates GFP transcription 7 araC ori pGLO bla GFP 3/14/2016 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. 8 3/14/2016 Work of the pGLO Plasmid Arabinose Operon RNA Polymerase Regulatory Gene--araC Binds RNA polymerase Gene products 3/14/2016 10 RNA Polymerase Transcriptional Regulation ara GFP Operon ara Operon araC B A D araC Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC 11 B A D GFP Gene GFP Gene RNA Polymerase araC GFP Gene 3/14/2016 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 12 3/14/2016 Transformation Procedures Day 1 Day 2 13 3/14/2016 Reasons for performing each transformation step? Ca++ O Transformation solution = CaCl2 Ca+2 Positive charge of ions shields negative charge of DNA phosphates 14 Ca++ O P O Base O CH2 O Sugar O Ca++ O P O Base O CH2 O Sugar OH 3/14/2016 Why perform each transformation step? Incubation on ice slows fluid cell membranes Cell wall GFP Heat-shock increases permeability of cell membrane Incubation in LB nutrient broth allows beta lactamase expression 15 Beta lactamase (ampicillin resistance) 3/14/2016 Inducible, Repressible or Both?? http://faculty.clintoncc.suny.edu/faculty/Mic hael.Gregory/files/bio%20101/Bio%20101 %20Lectures/Gene%20Regulation/gene.ht m 16 3/14/2016 The Gel Analysis 17 3/14/2016 Graphing Migration Distance 18 3/14/2016