Title: pGLO
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2pGLO Transformation and Purification of Green
Fluorescent Protein (GFP)
3Instructors
- 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
4Why TeachBacterial Transformationand Protein
Purification?
- Powerful teaching tool
- Laboratory extensions
- Real-world connections
- Link to careers and industry
- Standards based
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6pGLO 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!
7Green Fluorescent Protein (GFP) Chromatography Kit
- GFP Purification Kit Advantages
- Cloning in action
- Links to biomanufacturing
- Biopharmaceutical development
- Amazing visual results
8WorkshopTime Line
- Introduction
- Transform bacteria with pGLO plasmid
9Central Framework of Molecular Biology
10Links 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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12Using GFP as a biological tracer
http//www.conncoll.edu/ccacad/zimmer/GFP-ww/prash
er.html With permission from Marc Zimmer
13pGLO Bacterial Transformation Kit
14Transformation Procedure Overview
Day 1
10
15What is Transformation?
GFP
- Uptake of foreign DNA, often a circular plasmid
Beta-lactamase Ampicillin Resistance
16What 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
17Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
18The Many Faces of Plasmids
Graphic representation
Scanning electron micrograph of supercoiled
plasmid
19GeneExpression
- Beta Lactamase
- Ampicillin resistance
- Green Fluorescent Protein (GFP)
- Aequorea victoria jellyfish gene
- araC regulator protein
- Regulates GFP transcription
20Bacterial Transformation
Cell wall
GFP
Bacterial chromosomal DNA
Beta lactamase (ampicillin resistance)
pGLO plasmids
21Transcriptional Regulation
- Lactose operon
- Arabinose operon
- pGLO plasmid
22Transcriptional Regulation
23Gene Regulation
24Methods of Transformation
- Electroporation
- Electrical shock makes cell membranes permeable
to DNA - Calcium Chloride/Heat-Shock
- Chemically-competent cells uptake DNA after heat
shock
25Transformation 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
26Reasons 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
27Why 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)
28What is Nutrient Broth?
- Luria-Bertani (LB) broth
- Medium that contains nutrients for bacterial
growth and gene expression - Carbohydrates
- Amino acids
- Nucleotides
- Salts
- Vitamins
29Grow? Glow?
- Follow protocol
- On which plates will colonies grow?
- Which colonies will glow?
30LaboratoryQuick Guide
31Volume Measurement
32Webinars
- Enzyme Kinetics A Biofuels Case Study
- Real-Time PCR What You Need To Know and Why You
Should Teach It! - Proteins Where DNA Takes on Form and Function
- From plants to sequence a six week college
biology lab course - From singleplex to multiplex making the most out
of your realtime experiments - explorer.bio-rad.com?Support?Webinars