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pGLO ™ & GFP

pGLO ™ & GFP. Uses of Green Fluorescent Protein. Uses of GFP. 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

Samuel
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pGLO ™ & GFP

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  1. pGLO™ & GFP

  2. Uses of Green Fluorescent Protein Uses of GFP • 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

  3. Transformation is a natural process that Bacterial have evolved in order to obtain DNA from their environment. • Use of the procedure enables scientists to insert genes by recombinant techniques and place the plasmid into a bacteria for expression

  4. Transformation Procedure

  5. Timeline for Transformation • Background • Transform bacteria with pGLO plasmid • Purify GFP using column chromatography

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

  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 expressproteins of interest

  8. The Many Faces of Plasmids Scanning electron micrograph Graphic representation Agarose gel

  9. Plasmid Map • 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. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

  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 in 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 • Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH

  18. 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 Cell wall GFP 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?

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