pGLO

Report
Bacterial Transformation Lab
Procedure Overview
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Observe starter plate
Label blank tubes “+” and “-”
Pipet 250 µl TS into each tube  ice
Sterile loop 1 colony from starter into each tube
Sterile loop  plasmid into “+” tube (Mr. Mohn)
Ice for 10 min, label plates while waiting
Heat shock 50 sec  back to ice
Pipet 250 µl LB into each tube
Room temp for 10 min
Inoculate plates with 100 µl of “+” or “-”
Spread with sterile loop, stack and tape plates
Incubate 48 hours at 37 C
LB = Luria Broth
250 µl
100 µl
TS = Transformation Solution
What is Bacterial
Transformation?
• Bacterial cells
taking up DNA
from their
environment.
• Discovered by
Frederick
Griffith.
What is a plasmid?
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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
ori
bla
Bacterial Cells and DNA
Chromosomal
Bacterial cell
Plasmid DNA
Chromosomal DNA
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!
pGLO
• A fluorescent protein
from the jellyfish,
Aequorea victoria
• The plasmid used in this
lab contains the genes
necessary for producing
and expressing the
pGLO protein in
whatever organism it is
found in.
Other Glowing Critters
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
pGLO
bla
GFP
Green Fluorescent Protein
(GFP)
• Useful in research to track the fate of
specific cells during development.
• Other fluorescent proteins are now also
available for use in genetic research.
Transformation Results
Only cells
getting pGLO
plasmid grow
Only cells
getting pGLO
plasmid grow
and glow
Without pGLO
plasmid,
nothing can
grow
All cells grow
since there is
no antibiotic
on the plate

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