Download
slide1 n.
Skip this Video
Loading SlideShow in 5 Seconds..
pGLO TM Bacterial Transformation Courtesy BioRad Corporation PowerPoint Presentation
Download Presentation
pGLO TM Bacterial Transformation Courtesy BioRad Corporation

pGLO TM Bacterial Transformation Courtesy BioRad Corporation

433 Vues Download Presentation
Télécharger la présentation

pGLO TM Bacterial Transformation Courtesy BioRad Corporation

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. pGLOTM Bacterial Transformation Courtesy BioRad Corporation

  2. pGLO/ Green Fluorescent Protein IsolationA single bacteria might be worth 5 billion dollars to the right buyer.

  3. DNA RNA Protein Trait Central Framework of Molecular Biology

  4. What is transformation? • Uptake of foreign DNA, often a circular plasmid, from the surroundings with the expression of these genes. GFP Amp Resistance

  5. ori bla 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. Uses of Genetic Transformation • Agriculture • Introduction of drought, pest, or frost resistance genes “BT Corn” • Bioremediation • Introduction of genes into bacteria giving them the ability to degrade hydrocarbons, “Oil Eating Bacteria” • Medicine • Gene therapy • Production of human proteins • Insulin, clotting factors

  7. araC ori pGLO GFP bla pGLO Plasmid-Draw this!! • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription

  8. Purpose of pGLO Transformation • To transform E. coli K-12 with a plasmid that contains a gene that codes for Green Fluorescent Pigment (GFP) • Transformed cells will produce GFP and glow a brilliant green under UV light.

  9. Work of the pGLO Plasmid

  10. Arabinose Operon RNA Polymerase Regulatory Gene--araC Binds RNA polymerase Gene products RNA Polymerase

  11. ara GFP Operon araC GFP Gene araC GFP Gene RNA Polymerase araC GFP Gene Transcriptional Regulation ara Operon araC B A D Effector(Arabinose) Effector(Arabinose) araC B A D RNA Polymerase araC B A D

  12. Transformation Procedure • Suspend bacterial colonies in Transformation Solution • Add pGLO plasmid DNA • Place tubes on ice • Heat shock at 42oC and place on ice • Incubate withnutrient broth • Streak plates

  13. Transformation Procedures Day 1 Day 2

  14. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Reasons for performing each transformation step? • Transformation solution = CaCl2 Positive charge of Ca+2 ions shields negative charge of DNA phosphates

  15. Cell wall GFP Beta lactamase (ampicillin resistance) Why perform each transformation step? • Incubation on iceslows fluid cell membranes • Heat-shockincreases permeability of cell membrane • Incubation in LB nutrient brothallows beta lactamase expression

  16. Inducible, Repressible or Both?? http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/bio%20101/Bio%20101%20Lectures/Gene%20Regulation/gene.htm

  17. The Gel Analysis

  18. Graphing Migration Distance