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In the pGLO lab, we will:

In the pGLO lab, we will:. Use recombinant DNA Genetically engineer E. coli bacteria by inserting a plasmid Plate and grow bacteria Determine if the proteins are expressed by genes. Single celled Use plasmid DNA Reproduce quickly.

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In the pGLO lab, we will:

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  1. In the pGLO lab, we will: • Use recombinant DNA • Genetically engineer E. coli bacteria by inserting a plasmid • Plate and grow bacteria • Determine if the proteins are expressed by genes

  2. Single celled Use plasmid DNA Reproduce quickly Why are bacteria a good choice for genetic transformation?

  3. Source of “glowing gene” for this experimentAequorea victoria: jellyfish Gene isolated by restriction enzymes and placed into plasmid

  4. araC ori pGLO GFP bla pGLO Plasmid – 3 genes • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein • jellyfish gene – glows • green in prescence of • arabinose sugar • araC regulator protein • Control gene (switches on in the presence of arabinose) • Creates a protein that turns on GFP gene!

  5. DNA binding Protein: Represses Transcription Genes coding for digestive enzymes araC B A D Effector(Arabinose) Promotor(PBAD) araC B A D RNA Polymerase Transcription B A D araC Arabinose Operon

  6. GFP Gene araC Effector(Arabinose) Promotor (PBAD) araC GFP Gene RNA Polymerase Transcription araC GFP Gene Ara-GFP Operon • GFP only produced in the presence of Arabinose • Genes coding for digestive enzymes have been replaced by the GFP gene: no metabolism of arabinose

  7. Bacterial TransformationPlasmids enter bacterial cell and genes are expressed Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

  8. What is in the agar? • LB – nutrient broth for bacteria to feed on • Ampicillin – antibiotic that kills bacteria • Arabinose – Sugar necessary to switch on ara C gene and the GFP gene

  9. Transformation Procedure: Overview • Suspend bacterial colonies in Transformation Solution • Add pGLO plasmid DNA • Place tubes on ice • Heat shock at 42oC and place on ice • Incubate with LB nutrient broth • Streak plates

  10. Reasons for Each • Transformation Step: • CaCl2 treatment (TS) • Positive charge of Ca 2+ • neutralizes: • • negative charge of DNA • • negative charge of cell membrane

  11. 2.Incubation on ice slows cell membranes 3. Heat-shock Increases permeability of cell membrane These steps allow the plasmid to be taken in by the bacteria cells

  12. 4. Nutrient broth incubation allows beta lactamase gene to be expressed (for antibiotic resistance)

  13. If bacteria grows in the presence of ampicillin If bacteria glows in the presence of arabinose How will we know if bacteria is transformed?

  14. pGLO Lab • Purpose: To transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar and grow in the presence of the antibiotic, ampicillin.

  15. Sterile Technique • Bacteria are UBIQUITOUS…they are found EVERYWHERE! • Sterile technique refers to procedures that reduce the possibility of contamination…these techniques protect YOU, your CULTURES and REAGENTS, and LAB EQUIPMENT

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