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Transport

The Cell:. DNA. gene. Nucleus. mRNA. Transport. Cytoplasm. Protein. ori. bla. What is a plasmid?. Circular piece of autonomously replicating DNA. Originally evolved by bacteria May express antibiotic resistance or be modified to express proteins of interest. araC. ori. pGLO.

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Transport

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  1. The Cell: DNA gene Nucleus mRNA Transport Cytoplasm Protein

  2. ori bla What is a plasmid? • Circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance • or be modified to express proteins of interest

  3. araC ori pGLO GFP bla pGLO Plasmid • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription

  4. What is transformation? • Uptake of foreign DNAoften a circular plasmid GFP Amp Resistance

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

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

  7. Transformation Procedure • Suspend colonies in Transformation Solution • Add pGLO plasmid DNA to “+” tube • Incubate tubes 10 minutes on ice • Heat shock 50 seconds at 42oC • Incubate 2 minutes on ice • Incubate 10 minutes withLB broth • Plate

  8. Why perform each transformation step? 1. Incubation on ice slows fluid cell membranes 2. Heat-shock increases permeability of cell membrane 3. LB recovery allows beta lactamase expression Cell wall GFP Beta lactamase (ampicillin resistance)

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