Transcript pGLO ™ Transformation

pGLO and Ampicillin Resistance
Transformation of E. coli
Inspire your students – tomorrow’s researchers
CCTCA February 12, 2016
Ampicillin resistance
and pGLO™
Transformation
Bob Malyk BSc., MEd.
[email protected]
Biology Teacher (retired)
Ridley College
St. Catharines, ON
Canada
Bio-Rad (Canada) Life
Science Education –
Biotechnology Explorer
Consultant
GFP
Workshop
Timeline
• Introduction
• Transform bacteria with pGLO plasmid
Why Teach
Bacterial
Transformation
and Protein
Purification?
• Powerful teaching tool
• Meet national science standards
• Laboratory extensions
• Real-world connections
• Link to careers and industry
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
pGLO™
Bacterial
Transformation
Kit
Bio-Rad pGLO Kit Advantages
• Standards-based
• Comprehensive curricula for inquiry-based
investigations
• Compatible with 50 minute class periods
• Serves entire class of 32 students
(up to 4 students per group)
• Cost effective $120.80 ($3.78/student)
• Success in student’s hands
• Safe
• Striking results!
Transformation
Procedure
• Suspend bacterial colonies in
Transformation solution
• Add pGLO plasmid DNA
• Place tubes in ice
• Heat-shock at 42°C and place on ice
• Incubate with nutrient broth
• Streak plates
Transformation
Procedure
Day 1
Day 2
Volume
Measurement
pGLO Bacterial
Transformation
Kit
What is
Transformation?
• 1928 Frederick
Griffith
• 1944 Avery and
McLeod
• Uptake of foreign
DNA, often a circular
plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
What is
Transformation?
F. Griffith
1928
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
The Many
Faces of
Plasmids
Transmission electron micrograph
Graphic representation
Agarose gel
Protein Size
• 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
Bacterial
DNA
Bacterial cell
Plasmid DNA
Genomic DNA
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
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?