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pGLO™ & GFP

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pGLO & GFP pGLO Transformation and Purification of Green Fluorescent Protein (GFP) Instructors Stan Hitomi Director, Edward Teller Education Center UC Davis ... – PowerPoint PPT presentation

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Title: pGLO™ & GFP


1
(No Transcript)
2
pGLO GFP
3
pGLO Transformationand Purification of Green
Fluorescent Protein (GFP) Instructors
  • Stan Hitomi
  • Director, Edward Teller Education Center
  • UC Davis / Lawrence Livermore National
  • Laboratory, Livermore, CA
  • Kirk Brown
  • Lead Instructor, Edward Teller Education Center
  • Science Dept. Chair, Tracy High School
  • and Delta College, Tracy, CA
  • Sherri Andrews, Ph.D.
  • North Carolina School of the Arts
  • Winston-Salem, NC

4
Why Teach Bacterial Transformationand Protein
Purification?
  • Powerful teaching tool
  • Meet national science standards
  • Laboratory extensions
  • Real-world connections
  • Link to careers and industry

5
Central Framework of Molecular Biology
6
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

7
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!

8
Green Fluorescent Protein (GFP) Chromatography Kit
  • GFP Purification kit advantages
  • Links to real world science
  • Laboratory extensions
  • Tangible results
  • Leverages equipment
  • Amazing results

9
Transformation Procedure
Day 1
  • Day 2

10
GFP Workshop Timeline
  • Introduction
  • Transform bacteria with pGLO plasmid
  • Purify GFP using column chromatography

11
pGLO Bacterial Transformation Kit
12
What is Transformation?
  • Uptake of foreign DNA, often a circular plasmid

13
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

14
The Many Faces of Plasmids
Transmission electron micrograph
Agarose gel
Graphic representation
15
Protein Size
  • Beta Lactamase
  • Ampicillin resistance
  • Green Fluorescent Protein (GFP)
  • Aequorea victoria jellyfish gene
  • araC regulator protein
  • Regulates GFP transcription

16
Bacterial Transformation
Cell wall
GFP
Bacterial chromosomal DNA
Beta lactamase (ampicillin resistance)
pGLO plasmids
17
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
18
Transcriptional Regulation
  • Lactose operon
  • Arabinose operon
  • pGLO plasmid

19
Transcriptional Regulation
20
Gene Regulation
21
Methods of Transformation
  • Electroporation
  • Electrical shock makes cell membranes permeable
    to DNA
  • Calcium Chloride/Heat-Shock
  • Chemically-competent cells uptake DNA after heat
    shock

22
Transformation Procedure
  • Suspend bacterial colonies in Transformation
    solution
  • Add pGLO plasmid DNA
  • Place tubes in ice
  • Heat-shock at 42C and place on ice
  • Incubate with nutrient broth
  • Streak plates

23
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
24
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)
25
What is Nutrient Broth?
  • Luria-Bertani (LB) broth
  • Medium that contains nutrients for bacterial
    growth and gene expression
  • Carbohydrates
  • Amino acids
  • Nucleotides
  • Salts
  • Vitamins

26
Grow? Glow?
  • Follow protocol
  • On which plates will colonies grow?
  • Which colonies will glow?

27
Volume Measurement
28
GFP Chromatography Kit
29
GFP Purification Procedures
Day 1
Day 3
  • Day 2

30
Why Use Chromatography?
  • To purify a single recombinant protein of
    interest from over 4,000 naturally occuring E.
    coli gene products.

31
Column Chromatography
  • Chromatography used for protein purification
  • Size exclusion
  • Ion exchange
  • Hydrophobic interaction

32
HydrophobicInteractions
Hydrophobic bead
  • Aqueous solution hydrophobic
  • High salt hydrophobic

H
O
-
-
H

O
O
S
N
H
H
O
O
-
O
S
O
-
O
33
Hydrophobic Interaction ChromatographySteps 13
  • Add bacterial lysate to column matrix in
  • high salt buffer
  • 2. Wash less hydrophobic proteins from column in
    low salt buffer
  • 3. Elute GFP from column with no salt buffer

34
Step 1 Hydrophobic Interaction Chromatography
  • Add bacterial lysate to column matrix in high
    salt buffer
  • Hydrophobic proteins interact with column

35
Step 2 Hydrophobic Interaction Chromatography
  • Wash less hydrophobic from column with low salt
    buffer
  • Less hydrophobic E. coli proteins fall from
    column
  • GFP remains bound to the column

36
Step 3 Hydrophobic Interaction Chromatography
  • Elute GFP from column by adding no salt buffer
  • GFP
  • Released from column matrix
  • Flows through the column

37
Helpful Hints Hydrophobic Interaction
Chromatography
  • Add a small piece of paper to collection tube
    where column seats to insure column flow
  • Rest pipette tip on side of column to avoid
    column bed disturbance when adding solutions
  • Drain until the meniscus is just above the matrix
    for best separation
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