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pGLO ™ & GFP. Uses of Green Fluorescent Protein. Uses of GFP. 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

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Pglo gfp l.jpg
pGLO™ & GFP


Uses of gfp l.jpg

Uses of Green Fluorescent Protein

Uses of GFP

  • 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


Slide3 l.jpg


Transformation procedure l.jpg
Transformation evolved in order to obtain DNA from their environment.Procedure


Slide5 l.jpg

Timeline for Transformation evolved in order to obtain DNA from their environment.

  • Background

  • Transform bacteria with pGLO plasmid

  • Purify GFP using column chromatography


What is transformation l.jpg

GFP evolved in order to obtain DNA from their environment.

Beta-lactamase

Ampicillin

Resistance

What is Transformation?

  • Uptake of foreign DNA, often a circular plasmid


What is a plasmid l.jpg
What is a plasmid? evolved in order to obtain DNA from their environment.

  • A circular piece of autonomously replicating DNA

  • Originally evolved by bacteria

  • May express antibiotic resistance gene or be modified to expressproteins of interest


The many faces of plasmids l.jpg
The Many Faces of Plasmids evolved in order to obtain DNA from their environment.

Scanning electron micrograph

Graphic representation

Agarose gel


Plasmid map l.jpg
Plasmid Map evolved in order to obtain DNA from their environment.

  • Beta Lactamase

    • Ampicillin resistance

  • Green Fluorescent Protein (GFP)

    • Aequorea victoria jellyfish gene

  • araC regulator protein

    • Regulates GFP transcription


Bacterial transformation l.jpg
Bacterial Transformation evolved in order to obtain DNA from their environment.

Cell wall

GFP

Bacterial

chromosomal DNA

Beta lactamase

(ampicillin resistance)

pGLO plasmids


Bacterial dna l.jpg
Bacterial DNA evolved in order to obtain DNA from their environment.

Bacterial cell

Plasmid DNA

Genomic DNA


Transcriptional regulation l.jpg
Transcriptional Regulation evolved in order to obtain DNA from their environment.

  • Lactose operon

  • Arabinose operon

  • pGLO plasmid


Transcriptional regulation13 l.jpg

ara Operon evolved in order to obtain DNA from their environment.

lac Operon

araC

B

A

D

LacI

Z

Y

A

Effector (Arabinose)

Effector(Lactose)

araC

B

A

D

LacI

Z

Y

A

RNA Polymerase

RNA Polymerase

B

A

D

araC

Z

Y

A

Transcriptional Regulation


Gene regulation l.jpg

ara GFP Operon evolved in order to obtain DNA from their environment.

ara Operon

araC

GFP Gene

araC

B

A

D

Effector(Arabinose)

Effector (Arabinose)

araC

B

A

D

araC

GFP Gene

RNA Polymerase

RNA Polymerase

B

A

D

araC

araC

GFP Gene

Gene Regulation


Methods of transformation l.jpg
Methods of Transformation evolved in order to obtain DNA from their environment.

  • Electroporation

    • Electrical shock makes cell membranes permeable to DNA

  • Calcium Chloride/Heat-Shock

    • Chemically-competent cells uptake DNA after heat shock


Transformation procedure16 l.jpg
Transformation Procedure evolved in order to obtain DNA from their environment.

  • Suspend bacterial colonies in Transformation solution

  • Add pGLO plasmid DNA

  • Place tubes in ice

  • Heat-shock at 42°C and place on ice

  • Incubate with nutrient broth

  • Streak plates


Reasons for performing each transformation step l.jpg
Reasons for Performing Each Transformation Step? evolved in order to obtain DNA from their environment.

Ca++

O

Ca++

O

P

O

  • Transformation solution = CaCI2

    Positive charge of Ca++ ions shields negative

    charge of DNA phosphates

Base

O

O

CH2

Sugar

O

Ca++

O

O

P

Base

O

O

CH2

Sugar

OH


Why perform each transformation step l.jpg
Why Perform Each Transformation Step? evolved in order to obtain DNA from their environment.

2.Incubate on ice

slows fluid cell membrane

3. Heat-shock

Increases permeability of membranes

4. Nutrient broth incubation

Allows beta-lactamase

expression

Cell wall

GFP

Beta-lactamase

(ampicillin resistance)


What is nutrient broth l.jpg
What is Nutrient Broth? evolved in order to obtain DNA from their environment.

  • Luria-Bertani (LB) broth

  • Medium that contains nutrients for bacterial growth and gene expression

    • Carbohydrates

    • Amino acids

    • Nucleotides

    • Salts

    • Vitamins


Grow glow l.jpg

LB/Amp evolved in order to obtain DNA from their environment.

LB/Amp/Ara

LB

Grow?Glow?

  • Follow protocol

  • On which plates will colonies grow?

  • Which colonies will glow?


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