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pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP). DNA. RNA. Protein. Trait. Central Framework of Molecular Biology. GFP is a visual marker Study of biological processes (example: synthesis of proteins) Localization and regulation of gene expression Cell movement
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DNA RNA Protein Trait Central Framework of Molecular Biology
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 Links to Real-world
Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
Transformation:Procedure Overview Day 2 Day 1
What is Transformation? • Uptake of foreign DNA, often a circular plasmid
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
Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid
ara Operon lac Operon araC B A D LacI Z Y A Effector (Arabinose) Effector(Lactose) araC B A D LacI Z Y A RNA Polymerase RNA Polymerase B A D araC Z Y A Transcriptional Regulation
ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation
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 http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
Reasons for Performing Each Transformation Step? • Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates
Why Perform Each Transformation Step? 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression
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?
When transferring bacteria to or from an agar plate • Organisms are transferred by using a sterile loop and reaching in from the side while keeping the plate covered as much as possible. • This technique minimizes the risk of contamination from above. http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
When adding DNA to the transformation reaction • Immerse a sterile loop into the bottle containing plasmid DNA. • When the center of the loop is coated with a soap-like film, transfer it the + DNA microtube http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/