From a Gel to a Cell:

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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

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pGLO Plasmid

• Beta Lactamase
• Ampicillin resistance
• Green Fluorescent Protein
• Aequorea victoria jellyfish gene
• araC regulator protein
• Regulates GFP transcription

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What is transformation?

• Uptake of foreign DNA often a circular plasmid

GFP
Amp Resistance
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pGLO Transformation
Cell wall
GFP
Bacterial chromosomal DNA
Beta lactamase (ampicillin resistance)
pGLO plasmids
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pGLO Regulation
ara Operon
araC
B
A
D
Effector (Arabinose)
Effector (Arabinose)
araC
B
A
D
RNA Polymerase
B
A
D
araC
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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 with LB broth
• Plate

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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)