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pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP). Instructors. Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center

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pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

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  1. pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

  2. Instructors Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

  3. WhyTeachBacterial Transformationand Protein Purification? • Powerful teaching tool • Laboratory extensions • Real-world connections • Link to careers and industry • Standards based

  4. pGLO™ Bacterial Transformation Kit Bio-Rad pGLO Kit Advantages • Standards-based • Comprehensive curricula for inquiry-based investigations • Compatible with 50 minute class periods • Serves entire class of 32 students (up to 4 students per group) • Cost-effective • Success in student’s hands • Safe • Striking results!

  5. Green Fluorescent Protein (GFP) Chromatography Kit GFP Purification Kit Advantages • Cloning in action • Links to biomanufacturing • Biopharmaceutical development • Amazing visual results

  6. WorkshopTime Line • Introduction • Transform bacteria with pGLO plasmid

  7. DNA RNA Protein Trait Central Framework of Molecular Biology

  8. 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

  9. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

  10. pGLO Bacterial Transformation Kit

  11. Transformation Procedure Overview Day 2 Day 1 10

  12. What is Transformation? • Uptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance

  13. 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

  14. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

  15. The Many Faces of Plasmids Graphic representation Scanning electron micrograph of supercoiled plasmid

  16. GeneExpression • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein (GFP) • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription

  17. Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

  18. Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid

  19. 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

  20. 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

  21. Methods of Transformation • Electroporation • Electrical shock makes cell membranes permeable to DNA • Calcium Chloride/Heat-Shock • Chemically-competent cells uptake DNA after heat shock

  22. 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

  23. Reasons for Performing Each Transformation Step? Ca++ O Ca++ O P O Base • Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH

  24. 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)

  25. What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins

  26. LB/Amp LB/Amp/Ara LB Grow?Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow?

  27. LaboratoryQuick Guide

  28. Volume Measurement

  29. Webinars • Enzyme Kinetics — A Biofuels Case Study • Real-Time PCR — What You Need To Know and Why You Should Teach It! • Proteins — Where DNA Takes on Form and Function • From plants to sequence: a six week college biology lab course • From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars

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