pGLOTM Bacterial Transformation Courtesy BioRad Corporation 4/21/2017

pGLO/ Green Fluorescent Protein Isolation A single bacteria might be worth 5 billion dollars to the right buyer.

Central Framework of Molecular Biology DNA RNA Protein Trait 4/21/2017

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

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

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

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

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

Work of the pGLO Plasmid

Arabinose Operon RNA Polymerase Regulatory Gene--araC Gene products Binds RNA polymerase Gene products 4/21/2017 RNA Polymerase

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

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

Transformation Procedures Day 1 Day 2 4/21/2017

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

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

Inducible, Repressible or Both?? http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/bio%20101/Bio%20101%20Lectures/Gene%20Regulation/gene.htm 4/21/2017

The Gel Analysis 4/21/2017

Graphing Migration Distance 4/21/2017