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

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

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  1. pGLO™ Transformation and Purification of 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. What is Transformation? • Uptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance

  6. 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

  7. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

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

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

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

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

  12. 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

  13. 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

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

  15. 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

  16. 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)

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

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

  19. GFP Electrophoresis Extension • SDS PAGE sample preps are made from white and green colonies • Bacterial lysates are prepared in Laemmli buffer • Samples are loaded onto polyacrylamide gels LB/amp LB/amp/ara

  20. GFP Visualization-During & Post Electrophoresis M W G M W G M W G • Samples are electrophoresed • Fluorescent GFP can be visualized during electrophoresis • Coomassie stained gels allow for visualization of induced GFP proteins Fluorescent isoform Non-fluorescent isoform During Electrophoresis Post Electrophoresis Prestained bands + UV activated GFP Fluorescent bands Coomassie stained bands

  21. Why Use Chromatography? • To purify a single recombinant protein of interest from over 4,000 naturally occurring E. coli gene products.

  22. Column Chromatography • Chromatography used for protein purification • Size exclusion • Ion exchange • Hydrophobic interaction

  23. Hydrophobic Interaction Chromatography:(HIC)Steps 1–3 • Add bacterial lysate to column matrix in high salt buffer 2. Wash less hydrophobic proteins from column in low salt buffer • Elute GFP from column with no salt buffer

  24. Hydrophobic bead H H O O - - - - H H + + O O O O S S N N H H H H O O + + O O - - - - O O S S O O - - O O + + Step 1:Hydrophobic Interaction Chromatography • Add bacterial lysate to column matrix in high salt buffer • Hydrophobic proteins interact with column • Salt ions interact with the less hydrophobic proteins and H2O

  25. Hydrophobic bead H O - - + H O O S N H H O + + + + O - - - - - O S O - O + + + + Step 2:Hydrophobic Interaction Chromatography • Wash less hydrophobic from column with low salt buffer • Less hydrophobic E. coli proteins fall from column • GFP remains bound to the column

  26. + + + + + - - - - - + + + + + Step 3:Hydrophobic Interaction Chromatography Hydrophobic bead • Elute GFP from column by adding a no-salt buffer GFP • Released from column matrix • Flows through the column

  27. Helpful Hints:Hydrophobic Interaction Chromatography • Add a small piece of paper to collection tube where column seats to insure column flow • Rest pipet tip on side of column to avoid column bed disturbance when adding solutions • Drain until the meniscus is just above the matrix for best separation

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