pglo™ transformation - boston university 2 pglo/biotechnology...transformation solution •add...
TRANSCRIPT
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pGLO™ Transformation
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Workshop
Time Line
• Introduction
• Transform bacteria with pGLO plasmid
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Central
Framework of
Molecular
Biology
DNA RNA Protein Trait
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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
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Using GFP as a
biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
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pGLO Bacterial
Transformation
Kit
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Transformation
Procedure
Overview
Day 1
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What is
Transformation?
• Uptake of foreign
DNA, often a circular plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
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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
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Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
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The Many
Faces of
Plasmids
Scanning electron micrograph of
supercoiled plasmid
Graphic representation
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Gene
Expression
• Beta Lactamase
– Ampicillin resistance
• Green Fluorescent
Protein (GFP)
– Aequorea victoriajellyfish gene
• araC regulator
protein
– Regulates GFP transcription
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Bacterial
Transformation
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Bacterial
chromosomal
DNA
Cell wall
GFP
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Transcriptional
Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
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Transcriptional
Regulation
B A DaraC
B A Dara
C
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA Polymerase
Z Y A
Z Y ALacI
Effector (Lactose)
Z Y ALacI
lac Operon
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Gene
Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araCGFP Gene
Effector (Arabinose)
B A DaraC
B A Dara
C
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
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Methods of
Transformation
• Electroporation
– Electrical shock makes cell membranes permeable to DNA
• Calcium Chloride/Heat-Shock
– Chemically-competent cells uptake DNA after heat shock
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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
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Reasons for
Performing
Each
Transformation
Step?
1. Transformation
solution = CaCI2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++
Ca++
OCH
2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
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Why Perform
Each
Transformation
Step?
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)
Cell wall
GFP
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What is
Nutrient
Broth? • Luria-Bertani (LB) broth
• Medium that contains nutrients for
bacterial growth and gene expression
– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins
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Grow?
Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
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Laboratory
Quick Guide
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Volume
Measurement