Transformation and Protein Purification

1
pGLOTM Bacterial Transformation
Mr. Barber Hershey High School AP
Biology 2006-2007
2
Aequorea victoria
3
Why teach bacterial transformation and protein
purification?

• Powerful teaching tools
• Meet national science standard requirements
• Laboratory extensions
• Real-world connections
• Link to future careers and industry

4
Central Framework of Molecular Biology
5
Links to Real-world

• study of biological processes (ex. biosynthesis
  of proteins)
• localization of gene expression
• cell movement
• cell fate during development
• formation of different organs
• screenable marker to identify transgenic
  organisms

6
GFP Timeline

• Introduction
• Transform bacteria with pGLO plasmid
• Purify GFP using column chromatography

7
What is transformation?

• Uptake of foreign DNA, often a circular plasmid

GFP
Amp Resistance
8
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

9
The many faces of plasmids
Transmission electron micrograph
Agarose Gel
Graphic
10
pGLO Plasmid

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

11
Bacterial Transformation
12
Bacterial DNA
Genomic
Bacterial cell
Plasmid DNA
Genomic DNA
13
Transcriptional Regulation

• Lactose operon
• Arabinose operon
• pGLO plasmid

14
Transcriptional Regulation
RNA Polymerase
15
Gene Regulation
ara Operon
araC
B
A
D
Effector (Arabinose)
Effector (Arabinose)
araC
B
A
D
RNA Polymerase
B
A
D
araC
16
Methods of transformation

• Electroporation
• Electrical shock makes cell membranes permeable
  to DNA
• Calcium Chloride/Heat Shock
• Chemically-competent cells uptake DNA after heat
  shock

17
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

18
Reasons for performing each transformation step?

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

19
Why perform each transformation step?

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

E. coli Animation
20
What is nutrient broth?

• Luria-Bertani (LB) broth
• Medium that contains nutrients for bacterial
  growth and gene expression
• carbohydrates
• amino acids
• nucleotides
• salts
• vitamins

21
Grow? Glow?

• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?