bacterial transformation lab “pglo”. pglo a fluorescent protein from the jellyfish, aequorea...
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Bacterial Transformation Lab “pGLO”
pGLO
• A fluorescent protein from the jellyfish, Aequorea victoria
• The pGLO plasmid contains several genes that are necessary for producing and expressing the pGLO protein in whatever organism it is found in.
Other Glowing Critters
What is Bacterial Transformation?
• Bacterial cells taking up DNA from their environment.
• Discovered by Frederick Griffith.
Bacterial Transformation Lab
1. Bacterial Cells and plasmid DNA are mixed.
2. Cells take up plasmid.
3. Cell/DNA mix is plated on nutrient agar with antibiotic.
4. Only cells which obtained plasmid DNA will grow…and glow!
What is a plasmid?
ori
bla
• Small circular DNA molecule.
• Replicates autonomously • Originally evolved in
bacteria• May contain antibiotic
resistance gene or be modified to contain other genes.– bla is an ampicillin
resistance gene
Bacterial Cells and DNA
Chromosomal DNA
Chromosomal
Bacterial cell
Plasmid DNA
pGLO
blaGFP
pGLO Plasmid: Most Important
Components• bla gene– Bacteria with this gene
produce beta lactamase, an enzyme that allows them to grow in the presence of ampicillin
• GFP gene– Bacteria with this gene
glow under UV light
Reasons for Each Transformation Step
CaCl2 treatment
Positive charge of Ca2+ ions neutralizes: • negative charge of
DNA phosphates • negative charge of
membrane phospholipids
Ca++
Ca++
OCH2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
Incubation on iceslows fluid cell membranes
Heat-shockincreases permeability of cell membrane
Nutrient broth incubationallows beta lactamase expression prior to
exposure
Growth on agar containing arabinose
initiates transcription of GFP genes (modified operon)
Reasons for Each Transformation Step
Transformation Results
Only cells getting pGLO plasmid grow and glow
Only cells getting pGLO plasmid grow
Without pGLO plasmid, nothing can grow
All cells grow since there is no antibiotic on the plate