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pGLO ™ & GFP

pGLO ™ & 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) Localization and regulation of gene expression Cell movement Cell fate during development

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pGLO ™ & GFP

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

  5. 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

  6. The Many Faces of Plasmids Transmission electron micrograph Graphic representation Agarose gel

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

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

  9. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

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

  11. 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

  12. 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

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

  14. 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

  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. Transformation Procedure Day 2 Day 1

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