pglo gfp
Download
Skip this Video
Download Presentation
pGLO ™ & GFP

Loading in 2 Seconds...

play fullscreen
1 / 20

PGLO GFP - PowerPoint PPT Presentation


  • 490 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'PGLO GFP' - Samuel


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
uses of 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
  • Screenable marker to identify transgenic organisms
slide3
Transformation is a natural process that Bacterial have evolved in order to obtain DNA from their environment.
  • Use of the procedure enables scientists to insert genes by recombinant techniques and place the plasmid into a bacteria for expression
slide5

Timeline for Transformation

  • Background
  • Transform bacteria with pGLO plasmid
  • Purify GFP using column chromatography
what is transformation

GFP

Beta-lactamase

Ampicillin

Resistance

What is Transformation?
  • Uptake of foreign DNA, often a circular plasmid
what is a plasmid
What is a plasmid?
  • 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
The Many Faces of Plasmids

Scanning electron micrograph

Graphic representation

Agarose gel

plasmid map
Plasmid Map
  • Beta Lactamase
    • Ampicillin resistance
  • Green Fluorescent Protein (GFP)
    • Aequorea victoria jellyfish gene
  • araC regulator protein
    • Regulates GFP transcription
bacterial transformation
Bacterial Transformation

Cell wall

GFP

Bacterial

chromosomal DNA

Beta lactamase

(ampicillin resistance)

pGLO plasmids

bacterial dna
Bacterial DNA

Bacterial cell

Plasmid DNA

Genomic DNA

transcriptional regulation
Transcriptional Regulation
  • Lactose operon
  • Arabinose operon
  • pGLO plasmid
transcriptional regulation13

ara Operon

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

ara GFP Operon

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
Methods of Transformation
  • Electroporation
    • Electrical shock makes cell membranes permeable to DNA
  • Calcium Chloride/Heat-Shock
    • Chemically-competent cells uptake DNA after heat shock
transformation procedure16
Transformation Procedure
  • 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
Reasons for Performing Each Transformation Step?

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
Why Perform Each Transformation Step?

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

LB/Amp

LB/Amp/Ara

LB

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