pGLO

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pGLO Transformation and Purification of Green
Fluorescent Protein (GFP)
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Instructors

• Stan Hitomi
• Coordinator Math Science
• Principal Alamo School
• San Ramon Valley Unified School District
• Danville, CA
• Kirk Brown
• Lead Instructor, Edward Teller Education Center
• Science Chair, Tracy High School
• and Delta College, Tracy, CA
• Bio-Rad Curriculum and Training Specialists
• Sherri Andrews, Ph.D.
• sherri_andrews_at_bio-rad.com
• Leigh Brown, M.A.
• leigh_brown_at_bio-rad.com

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Why TeachBacterial Transformationand Protein
Purification?

• Powerful teaching tool
• Laboratory extensions
• Real-world connections
• Link to careers and industry
• Standards based

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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
• Success in students hands
• Safe
• Striking results!

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Green Fluorescent Protein (GFP) Chromatography Kit

• GFP Purification Kit Advantages
• Cloning in action
• Links to biomanufacturing
• Biopharmaceutical development
• Amazing visual results

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WorkshopTime Line

• Introduction
• Transform bacteria with pGLO plasmid

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Central Framework of Molecular Biology
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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/prash
er.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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• Day 2

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What is Transformation?
GFP

• Uptake of foreign DNA, often a circular plasmid

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
Bacterial cell
Plasmid DNA
Genomic DNA
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The Many Faces of Plasmids
Graphic representation
Scanning electron micrograph of supercoiled
plasmid
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GeneExpression

• Beta Lactamase
• Ampicillin resistance
• Green Fluorescent Protein (GFP)
• Aequorea victoria jellyfish gene
• araC regulator protein
• Regulates GFP transcription

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Bacterial Transformation
Cell wall
GFP
Bacterial chromosomal DNA
Beta lactamase (ampicillin resistance)
pGLO plasmids
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Transcriptional Regulation

• Lactose operon
• Arabinose operon
• pGLO plasmid

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Transcriptional Regulation
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Gene Regulation
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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 42C and place on ice
• Incubate with nutrient broth
• Streak plates

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Reasons for Performing Each Transformation Step?
Ca
O
Ca
P
O
O
Base
O
O
CH2
Sugar

• Transformation solution CaCI2
• Positive charge of Ca ions shields negative
• charge of DNA phosphates

O
Ca
P
O
O
Base
O
O
CH2
Sugar
OH
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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)
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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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LaboratoryQuick Guide
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Volume Measurement
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