What is next after pGLO

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(No Transcript)
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What is next after pGLO bacterial
transformation?
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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
• Essy Levy, M.Sc.
• essy_levy_at_bio-rad.com
• Leigh Brown, M.A.

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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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WorkshopTime Line

• Introduction
• Background on GFP
• Protein Electrophoresis of GFP
• Purify GFP using column chromatography

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Starting PointTransformation Procedure Overview
Day 1

• Day 2

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Discovery of GFP

• Originally Isolated from the jellyfish Aequorea
  victoria
• Naturally occurring in many bioluminescent jelly
  fish, reef corals and marine crustaceans
• Recombinant GFP has 239 amino acids
• Expressed as a 26,870 Dalton protein
• Barrel structure with the fluorescent chromophore
  at center of the protein
• Nobel prize-winning molecule!

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What is a chromophore?A group of atoms and
electrons forming part of an organic molecule
that causes it to be colored
The GFP chromophore is comprised of three
adjacent amino acids.These amino acids are
enzymatically converted to an active cyclic
chromophore
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GFP ChromophoreAbsorbs at 395 nmEmits
at 509 nm

• In vivo, GFP complexes with aequorin, a
  calcium-binding protein which transfers energy,
  to excite GFP
• In vitro, UV light is used to excite the GFP
  chromophore, absorbing light at a wavelength of
  395 nm, and emitting at a longer wavelength of
  509 nm visible fluorescent green light

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Recombinant GFP
GFP has been mutated

• Increased fluorescent photostability
• Improved hydrophilicity
• Increased solubility
• Improved fluorescence
• Various colors available
• Improved use as a reporter protein

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Using GFP as a biological tracer or reporter
protein
http//www.conncoll.edu/ccacad/zimmer/GFP-ww/prash
er.html With permission from Marc Zimmer
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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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www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
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Protein Electrophoresis of GFP ProceduresOvervi
ew
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Students will learn Protein Electrophoresis of
GFP

• Prepare an SDS-PAGE sample and understand the
  components of Laemmli buffer
• Understand protein structure and mechanisms for
  protein folding and unfolding and how different
  conformations can be identified using
  electrophoresis
• Understand how proteins are separated during
  gel electrophoresis
• Understand the use of electrophoresis in the
  process of transformation to protein expression
• Understand chromophores and the basis of
  protein fluorescence
• Construct a standard curve and determine the
  molecular weight (MW) of an unknown protein

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Sample PreparationSDS-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)no heat samples
Labeled tubes1. White, no heat2. Green,
no heat3. White, heat4. Green, heatSamples
should be a healthy scoop of colonies

• Label four screw-capped microtubes
• Add 300 µl of Sample buffer to the two no heat
  tubes
• Using the inoculation loop, scrape a sample
  (20-100 colonies) from an LB/amp (white colonies)
  plate and transfer to the White, no heat tube
  and mix thoroughly
• Using the inoculation loop, scrape a sample
  (20-100 colonies) from an LB/amp/ara (green
  colonies) plate and transfer to the Green, no
  heat tube and mix thoroughly

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Sample Preparation SDS-PAGEheat samples

• Transfer 150 µl of the White, no heat mixture
  to the White,heat tube
• Transfer 150 µl of the Green, no heat mixture
  to the Green,heat tube
• Heat the heat tubes to 95C for 5 min in a
  water bath. Cool to room temperature
• There is an important link between the STRUCTURE
  and FUNCTION of the protein

No heating Heating
intact chromophore denatured chromophore
functional protein non-functional protein
fluorescent protein non-fluorescent protein
Heating the samples denatures the proteins
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What is in the Laemmli sample buffer?

• Tris buffer
• provides appropriate pH
• SDS (sodium dodecyl sulfate)
• Solubilizes and denatures proteins
• Adds a negative charge to the protein
• DTT
• (1,4-Dithiothreitol) reduces disulfide bonds, to
  help unfold proteins and protein complexes
• Glycerol
• Increases the density of the samples to help
  samples sink into wells of the gel
• Bromophenol Blue
• dye to visualize samples

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Load and electrophorese samples30min at
200Vin 1XTGS Buffer
UV illumination Coomassie Stain
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How Does SDS-PAGE Work?

• Denatures proteins using detergent, DTT, and heat
• Separates proteins based on size
• Negatively charged proteins move to positive
  electrode
• Smaller proteins move faster through the gel

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Why Use Polyacrylamide Gels to Separate Proteins?

• Polyacrylamide gel has a tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
• Average amino acid 110 daltons
• Average nucleotide pair 649 daltons
• 1 kilobase of DNA 650 kD
• 1 kilobase of DNA encodes 333 amino acids 36 kD

• Size measured in kilodaltons (kD)
• Dalton mass of hydrogen molecule
• 1.66 x 10-24 gram
• Average amino acid 110 daltons

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GFP Visualization-During Post Electrophoresis
During Electrophoresis
Post Electrophoresis

• Fluorescent GFP can be visualized during
  electrophoresis
• Coomassie stained gels allow for visualization of
  induced GFP proteins

Fluorescent isoform
Non- fluorescent isoform
Prestained bands UV activated GFP
Coomassie stained bands
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Determining the molecular weights of GFPs in
different conformations
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GFP Chromatography Kit
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GFP Purification Procedures Overview
Day 1
Day 3

• Day 2

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Why Use Chromatography?

• To purify a single recombinant protein of
  interest from over 4,000 naturally occurring E.
  coli gene products.

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Column Chromatography

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

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Hydrophobic Interaction Chromatography(HIC)Ste
ps 13

• Add bacterial lysate to column matrix in
• high salt buffer
• 2. Wash less hydrophobic proteins from column in
  low salt buffer
• Elute GFP from column with
• no salt buffer

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Step 1 Hydrophobic Interaction Chromatography

• Add bacterial lysate to column matrix in high
  salt buffer
• Hydrophobic proteins interact with column
• Salt ions interact with the less hydrophobic
  proteins and H2O

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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

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Step 3 Hydrophobic Interaction Chromatography
Hydrophobic bead

• Elute GFP from column by adding a
• no-salt buffer
• GFP
• Released from column matrix
• Flows through the column

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LaboratoryQuick Guide
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Helpful Hints Hydrophobic Interaction
Chromatography

• Add a small piece of paper to collection tube
  where column seats to insure column flow

• Rest pipet 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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Column Chromatography vs. SDS-PAGEfor protein
isolation and analysis
Both methods separate proteins from a complex mixture
Column Chromatography
SDS PAGE
Used to isolate a protein from a complex mixture of molecules based on its physical and/or chemical properties
HIC separates molecules based on hydrophobicity
Need to lyse open the cells to run the soluble proteins over the column
Very dilute concentration of GFP in the HIC column fractions
An analytical technique used to detect the presence of a protein of interest
Separates molecules based on size
Can compare denatured and intact proteins to study protein structure
High concentration of GFP from whole cell lysate samples (dense colonies)
Both techniques, used in concert can help scientists purify and analytically study proteins
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Transformationis only the beginning More
techniques are necessary to fully understand the
structure and nature of a protein.
pGLO Transformation

Protein Purification
Results may lead to more experiments!
Size and structure determination
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