pGLO

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pGLO GFP
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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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Transformation Procedure
Day 1

• Day 2

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What is Transformation?

• Uptake of foreign DNA, often a circular plasmid

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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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Protein Size

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

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Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
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Prokaryotes

• Gene expression can be either
• Constitutive- Gene always expressed.
• Induced- Expressed in response to
• environmental signal.
• Repressed- Expression shut off when
• environmental signal present.

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Lactose Operon

• Inducer -- lactose
• Absence
• Active repressor
• No expression

• Presence
• Inactivation of repressor
• Expression

• Negative control

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Repressor binds tooperator andprevents
structural geneexpessionunless inducer
ispresent.
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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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Some animations to illustrate the lac operon.

• Operon animation

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

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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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Skin, Eyes and Organs give off an eerie
lightOnly fur does not glow
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Column Chromatography

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

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GFP Purification Procedures
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 occuring E.
  coli gene products.

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HydrophobicInteractions
Hydrophobic bead

• Aqueous solution hydrophobic
• High salt hydrophobic

H
O
-
-
H

O
O
S
N
H
H
O
O
-
O
S
O
-
O
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Green FluorescentProtein
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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

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Step 1 Hydrophobic Interaction Chromatography

• Add bacterial lysate to column matrix in high
  salt buffer
• Hydrophobic proteins interact with column

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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

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

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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 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