Download pGLO™ Transformation and Purification of Green Fluorescent

pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)
Central
Framework of
Molecular
Biology
DNA
RNA
Protein
Trait
Links to
Real-world
• GFP is a visual marker
• Study of biological processes
(example: synthesis of proteins)
• Localization and regulation of gene
expression
• Cell movement
• Cell fate during development
• Formation of different organs
• Screenable marker to identify transgenic
organisms
Using GFP as a
biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
pGLO Bacterial
Transformation
Kit
Volume
Measurement
Sterile
Technique
• Work surfaces are decontaminated each
day and after viable spills
• All solid wastes are decontaminated before
disposal
• Wash hands…
– After handing organisms with recombinant DNA
– Before exiting lab
• Wear protective eyewear and gloves
Disposing
Biological
Materials
UV Lamp &
Goggles
• Soak lab tools in 10% bleach solution for at
least 20 minutes:
– Pipets
– Inoculating loops
– Items in contact with agar surface
Plasmid
Transfer
• Look carefully at the loop to see if there is
a film of plasmid solution across ring.
• No plasmid, no transformation, no glow!
Transformants
• When transferring transformed cells, use
great care
• Bacteria will quickly settle to the bottom of
the tube
• Make sure tube is closed and flick bottom
with your finger to agitate (stir) suspension
before withdrawing cells for plating (step
#10)
• Again, no cells on Petri Dish = no glow!
Innoculating
Petri Dish
• Be sure to spread bacteria by
“skating” the innoculating loop
across surface of Petri Dish
• Don’t ever dig or penetrate
surface of agar.
– This would allow colonies to go
“underground”
• Spread in multiple directions
Transformation
Procedure
Overview
Day 1
Day 2
What is
Transformation?
• Uptake of foreign
DNA, often a circular
plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
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
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
The Many
Faces of
Plasmids
Graphic representation
Scanning electron micrograph of
supercoiled plasmid
Gene
Expression
• Beta Lactamase
– Ampicillin resistance
• Green Fluorescent
Protein (GFP)
– Aequorea victoria
jellyfish gene
• araC regulator
protein
– Regulates GFP
transcription
Bacterial
Transformation
Cell wall
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Transcriptional
Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
Transcriptional
Regulation
ara Operon
lac Operon
LacI
Z
Y A
ara
C
Z
Y A
araC
Y A
B A D
RNA Polymerase
RNA Polymerase
Z
A D
Effector (Arabinose)
Effector (Lactose)
LacI
B
araC
B A D
Gene
Regulation
ara GFP Operon
ara Operon
ara
C
B
A D
araC
Effector (Arabinose)
Effector (Arabinose)
araC
B A D
araC
RNA Polymerase
araC
B A D
GFP Gene
GFP Gene
RNA Polymerase
araC
GFP Gene
Methods of
Transformation
• Electroporation
– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock
– Chemically-competent cells uptake DNA after
heat shock
Transformation
Procedure
• Suspend bacterial colonies in
Transformation solution
• Add pGLO plasmid DNA
• Place tubes on ice
• Heat-shock at 42°C and place on ice
• Incubate with nutrient broth
• Streak plates
Reasons for
Performing
Each
Transformation
Step?
Ca++
Ca++
O
O P O
O
CH2
Base
O
Sugar
1. Transformation
solution = CaCI2
Positive charge of
Ca++ ions shields
negative
charge of DNA
phosphates
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
Why Perform
Each
Transformation
Step?
Cell wall
GFP
2. Incubate on ice
slows fluid cell
membrane
3. Heat-shock
Increases permeability
of membranes
4. Nutrient broth
incubation
Allows beta-lactamase
expression
Beta-lactamase
(ampicillin
resistance)
What is
Nutrient
Broth?
• Luria-Bertani (LB) broth
• Medium that contains nutrients for
bacterial growth and gene expression
– Carbohydrates
– Amino acids
– Nucleotides
– Salts
– Vitamins
Grow?
Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
Laboratory
Quick Guide