Transcript pGLO
Bacterial Transformation Lab “pGLO” pGLO • A fluorescent protein from the jellyfish, Aequorea victoria • The pGLO plasmid contains several genes that are necessary for producing and expressing the pGLO protein in whatever organism it is found in. Other Glowing Critters What is Bacterial Transformation? • Bacterial cells taking up DNA from their environment. • Discovered by Frederick Griffith. Bacterial Transformation Lab 1. Bacterial Cells and plasmid DNA are mixed. 2. Cells take up plasmid. 3. Cell/DNA mix is plated on nutrient agar with antibiotic. 4. Only cells which obtained plasmid DNA will grow…and glow! What is a plasmid? • • • • Small circular DNA molecule. Replicates autonomously Originally evolved in bacteria May contain antibiotic resistance gene or be modified to contain other genes. – bla is an ampicillin resistance gene ori bla Bacterial Cells and DNA Chromosomal Bacterial cell Plasmid DNA Chromosomal DNA pGLO Plasmid: Most Important Components • bla gene – Bacteria with this gene produce beta lactamase, an enzyme that allows them to grow in the presence of ampicillin • GFP gene – Bacteria with this gene glow under UV light pGLO bla GFP Reasons for Each Transformation Step CaCl2 treatment Ca++ O Ca++ O P O Positive charge of Ca2+ ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids Base O O CH2 Sugar O Ca++ O P O Base O CH2 O Sugar OH Reasons for Each Transformation Step Incubation on ice slows fluid cell membranes Heat-shock increases permeability of cell membrane Nutrient broth incubation allows beta lactamase expression prior to exposure Growth on agar containing arabinose initiates transcription of GFP genes (modified operon) Transformation Results Only cells getting pGLO plasmid grow Only cells getting pGLO plasmid grow and glow Without pGLO plasmid, nothing can grow All cells grow since there is no antibiotic on the plate