pGLOTM Bacterial Transformation Courtesy BioRad Corporation
pGLO/ Green Fluorescent Protein IsolationA single bacteria might be worth 5 billion dollars to the right buyer.
DNA RNA Protein Trait Central Framework of Molecular Biology
What is transformation? • Uptake of foreign DNA, often a circular plasmid, from the surroundings with the expression of these genes. GFP Amp Resistance
ori bla 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
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
araC ori pGLO GFP bla pGLO Plasmid-Draw this!! • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription
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.
Arabinose Operon RNA Polymerase Regulatory Gene--araC Binds RNA polymerase Gene products RNA Polymerase
ara GFP Operon araC GFP Gene araC GFP Gene RNA Polymerase araC GFP Gene Transcriptional Regulation ara Operon araC B A D Effector(Arabinose) Effector(Arabinose) araC B A D RNA Polymerase araC B A D
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 withnutrient broth • Streak plates
Transformation Procedures Day 1 Day 2
Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Reasons for performing each transformation step? • Transformation solution = CaCl2 Positive charge of Ca+2 ions shields negative charge of DNA phosphates
Cell wall GFP Beta lactamase (ampicillin resistance) Why perform each transformation step? • Incubation on iceslows fluid cell membranes • Heat-shockincreases permeability of cell membrane • Incubation in LB nutrient brothallows beta lactamase expression
Inducible, Repressible or Both?? http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/bio%20101/Bio%20101%20Lectures/Gene%20Regulation/gene.htm