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Transformation of E. coli with Green Fluorescent Protein (GFP)

Transformation of E. coli with Green Fluorescent Protein (GFP). DNA. RNA. Protein. Trait. Central Framework of Molecular Biology. Links to Real-world. GFP is a visual marker Study of biological processes (example: synthesis of proteins)

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Transformation of E. coli with Green Fluorescent Protein (GFP)

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  1. Transformation of E. coli with Green Fluorescent Protein (GFP)

  2. DNA RNA Protein Trait Central Framework of Molecular Biology

  3. 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

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

  5. Transformation Procedure Overview Day 2 Day 1

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

  7. 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

  8. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

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

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

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

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

  13. 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

  14. 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

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

  16. 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

  17. 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

  18. 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)

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

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

  21. LaboratoryQuick Guide

  22. Volume Measurement

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