Download pGLO

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
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.
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?
•
•
•
•
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
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
Reasons for Each
Transformation Step
 CaCl2 treatment
Ca++
O
Ca++
O P O
Positive charge of Ca2+
ions neutralizes:
• negative charge of DNA
phosphates
• negative charge of
membrane
phospholipids
Base
O
O
CH2
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
Reasons for Each
Transformation Step
 Incubation on ice
slows fluid cell membranes
 Heat-shock
increases permeability of cell membrane
 Nutrient broth incubation
allows beta lactamase expression prior to exposure
 Growth on agar containing arabinose
initiates transcription of GFP genes (modified operon)
LB = Luria Broth
250 µl
100 µl
TS = Transformation Solution
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