Transformation and Protein Purification

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pGLOTM Bacterial Transformation
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Central Framework of Molecular Biology
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What is transformation?

• Uptake of foreign DNA, often a circular plasmid,
  from the surroundings with the expression of
  these genes.

GFP
Amp Resistance
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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

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Uses of Genetic Transformation

• Agriculture
• Introduction of drought, pest, or frost
  resistance genes
• Bioremediation
• Introduction of genes into bacteria giving them
  the ability to degrade hydrocarbons
• Medicine
• Gene therapy
• Production of human proteins
• Insulin, clotting factors

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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.

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Aequorea victoria
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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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Bacterial Transformation
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Transcriptional 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 bacterial colonies in Transformation
  Solution
• Add pGLO plasmid DNA
• Place tubes on ice
• Heat shock at 42oC and place on ice
• Incubate with nutrient broth
• Streak plates

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Transformation
Procedures
Day 1
Day 2
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Reasons for performing each transformation step?

• Transformation solution CaCl2
• Positive charge of Ca2 ions shields negative
  charge of DNA phosphates

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Why perform each transformation step?

• Incubation on ice slows fluid cell membranes
• Heat-shock increases permeability of cell
  membrane
• Incubation in LB nutrient broth allows beta
  lactamase expression