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pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP) Central Framework of Molecular Biology DNA RNA Protein Trait Links to Real-world • GFP is a visual marker • Study of biological processes (example: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer pGLO Bacterial Transformation Kit Volume Measurement Sterile Technique • Work surfaces are decontaminated each day and after viable spills • All solid wastes are decontaminated before disposal • Wash hands… – After handing organisms with recombinant DNA – Before exiting lab • Wear protective eyewear and gloves Disposing Biological Materials UV Lamp & Goggles • Soak lab tools in 10% bleach solution for at least 20 minutes: – Pipets – Inoculating loops – Items in contact with agar surface Plasmid Transfer • Look carefully at the loop to see if there is a film of plasmid solution across ring. • No plasmid, no transformation, no glow! Transformants • When transferring transformed cells, use great care • Bacteria will quickly settle to the bottom of the tube • Make sure tube is closed and flick bottom with your finger to agitate (stir) suspension before withdrawing cells for plating (step #10) • Again, no cells on Petri Dish = no glow! Innoculating Petri Dish • Be sure to spread bacteria by “skating” the innoculating loop across surface of Petri Dish • Don’t ever dig or penetrate surface of agar. – This would allow colonies to go “underground” • Spread in multiple directions Transformation Procedure Overview Day 1 Day 2 What is Transformation? • Uptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance 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 Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA The Many Faces of Plasmids Graphic representation Scanning electron micrograph of supercoiled plasmid Gene Expression • Beta Lactamase – Ampicillin resistance • Green Fluorescent Protein (GFP) – Aequorea victoria jellyfish gene • araC regulator protein – Regulates GFP transcription Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid Transcriptional Regulation ara Operon lac Operon LacI Z Y A ara C Z Y A araC Y A B A D RNA Polymerase RNA Polymerase Z A D Effector (Arabinose) Effector (Lactose) LacI B araC B A D Gene Regulation ara GFP Operon ara Operon ara C B A D araC Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC B A D GFP Gene GFP Gene RNA Polymerase araC GFP Gene Methods of Transformation • Electroporation – Electrical shock makes cell membranes permeable to DNA • Calcium Chloride/Heat-Shock – Chemically-competent cells uptake DNA after heat shock Transformation Procedure • Suspend bacterial colonies in Transformation solution • Add pGLO plasmid DNA • Place tubes on ice • Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates Reasons for Performing Each Transformation Step? Ca++ Ca++ O O P O O CH2 Base O Sugar 1. Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O Base O CH2 O Sugar OH Why Perform Each Transformation Step? Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance) What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression – Carbohydrates – Amino acids – Nucleotides – Salts – Vitamins Grow? Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow? Laboratory Quick Guide