Transcript Bio-Rad pGLO PowerPoint

pGLO Bacterial Transformation
Why teach transformation?
• Powerful teaching tool
Dramatic result!
• Contemporary science
• Easy and safe to do!
• Simple to set up
• Links to industry and careers
pGLO Bacterial Transformation
Advanced preparation
• Prepare agar plates
–1 hour
–3-7 days prior
• Prepare starter plates/aliquot solutions
–30 minutes
–24-36h prior
Freeze dried for shipping and storage
What is transformation?
• Uptake of foreign DNA
• often a circular plasmid
GFP
Amp Resistance
Genomic
Bacterial cell
Plasmid DNA
Genomic DNA
What is a plasmid?
• Circular piece of autonomously
replicating DNA
• Originally evolved by bacteria
• May be modified to express
proteins of interest
Aequorea victoria
Equipment needed
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pGLO Bacterial Transformation kit
UV lamp
Water Bath
Ice
Autoclave/pressure cooker
Health & safety
• E.coli
–K12 HB101
• GMO
–Autoclave waste
All volumes can be measured
with drop pipette or loop
Volume Measurement
Squeeze bulb before going into liquid!
Prepare tubes
• Label 2 tubes
– + pGLO
– - pGLO
• Add 250ml Transformation
Solution (T) to each and place
on ice
Positive Ca2+ ions shield
DNA phosphates
Add bacteria & plasmid
• Add small number of
bacteria and place
on ice
Ice slows fluid
membrane
movement
Add loop of pGLO plasmid
DNA to “+” tube.
Incubate on ice for 10 minutes.
pGLO Plasmid
araC
–Origin of replication (ori)
–Beta Lactamase (bla)
–Green Fluorescent Protein (GFP)
–Regulatory protein (araC)
ori
pGLO
bla
GFP
How many plasmid copies?
–Origin of replication (ori)
ori
pGLO
How to identify
transformants?
Beta Lactamase (bla)
Ampicillin resistance
pGLO
bla
GFP gene
–Green Fluorescent Protein (GFP)
pGLO
GFP
The very first GFP
transformation…
Martin Chalfie
Columbia University
GFP is a marker gene
Single cell labelling
C. Elegans, “The Worm”
Neuron
transformed with
GFP
Label plates
Label 4 plates:
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–
–
–
LB - pGLO
LB/amp - pGLO
LB/amp + pGLO
LB/amp/ara + pGLO
LB = Luria Bertani broth
Amp = ampicilin
Ara = arabinose
Heat shock !!!
50 seconds at 42oC
Increases membrane permeability
Then 2 minutes on ice
Add LB broth
Add 250µl LB broth and
incubate 10 minutes room
temperature
Nutrient recovery allows Beta
Lactamase (bla) expression
GFP gene regulation
–Regulatory protein (araC)
araC
pGLO
Arabinose
induces the
GFP gene
Lac operon
Nobel Prize 1965
• Francois Jacob
• Jacques Monod
• Andre Lwoff
GFP Gene Regulation
ara Operon
araC
B
A
D
pGLO plasmid
araC
Arabinose
araC
B
A
D
Arabinose
araC
RNA Polymerase
araC
B
A
D
GFP Gene
GFP Gene
RNA Polymerase
araC
GFP Gene
Biolistics
Electroporation
Other transformation methods
-pGLO results
• LB - pGLO
• LB/amp - pGLO
+pGLO results
• LB/amp + pGLO
• LB/amp/ara + pGLO
A “real”
set of
results
Handheld UV light for viewing results
Induced and non-induced
transformants
Plate out
• Pipette 100ml culture onto plates
• Spread with loop
• Tape plates shut
• Incubate
–37ºC overnight
–OR 30ºC 2 days
–OR 21ºC 3 days
Extension ideas
Purify that GFP protein!
Conclusion
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Dramatic result
Quick and safe
No specialist or expensive equipment
Contemporary science