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pGLO

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... gene expression Cell movement Cell fate during development Formation of different organs Screenable marker to identify transgenic organisms What is Transformation ... – PowerPoint PPT presentation

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Title: pGLO


1
pGLO GFP
2
Central Framework of Molecular Biology
3
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

4
What is Transformation?
  • Uptake of foreign DNA, often a circular plasmid

5
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

6
The Many Faces of Plasmids
Transmission electron micrograph
Agarose gel
Graphic representation
7
Protein Size
  • Beta Lactamase
  • Ampicillin resistance
  • Green Fluorescent Protein (GFP)
  • Aequorea victoria jellyfish gene
  • araC regulator protein
  • Regulates GFP transcription

8
Bacterial Transformation
Cell wall
GFP
Bacterial chromosomal DNA
Beta lactamase (ampicillin resistance)
pGLO plasmids
9
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
10
Transcriptional Regulation
  • Lactose operon
  • Arabinose operon
  • pGLO plasmid

11
Transcriptional Regulation
12
Gene Regulation
13
Methods of Transformation
  • Electroporation
  • Electrical shock makes cell membranes permeable
    to DNA
  • Calcium Chloride/Heat-Shock
  • Chemically-competent cells uptake DNA after heat
    shock

14
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

15
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
16
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)
17
What is Nutrient Broth?
  • Luria-Bertani (LB) broth
  • Medium that contains nutrients for bacterial
    growth and gene expression
  • Carbohydrates
  • Amino acids
  • Nucleotides
  • Salts
  • Vitamins

18
Grow? Glow?
  • Follow protocol
  • On which plates will colonies grow?
  • Which colonies will glow?

19
Transformation Procedure
Day 1
  • Day 2
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