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GFP Transformation Lab. Images taken without permission from http://upload.wikimedia.org/wikipedia/de/4/4d/Protein_GFP_1EMA.png , http://bioinfo.biotec.or.th/Picture/Cell%20Tutorial/image005.jpg , http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Jellyfish.jpeg.

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gfp transformation lab
GFP Transformation Lab

Images taken without permission from http://upload.wikimedia.org/wikipedia/de/4/4d/Protein_GFP_1EMA.png, http://bioinfo.biotec.or.th/Picture/Cell%20Tutorial/image005.jpg, http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Jellyfish.jpeg

slide2

Plasmid containing gene of interest

Protein we want to produce

Overall Goal of Lab Experiment

  • Use genetic engineering techniques to insert the GFP gene into E. coli
gfp green fluorescent protein
GFP (Green Fluorescent Protein)
  • Naturally produced in Jellyfish– Aequorea victoria
  • Discovered in 1960’s
  • Source of bioluminescence when exposed to UV light

Img Src: http://icbxs.ethz.ch/members/leu/jellyfish.gif , http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Jellyfish.jpeg

slide4

Structure of the GFP Protein

Img Src: http://wwwchem.leidenuniv.nl/metprot/armand/images/029l.jpg

why is bioluminescence useful in nature
Why Is Bioluminescence Useful in Nature?
  • Attract Mates
  • See Food
  • Defense

Img Src: http://www.sio.ucsd.edu/explorations/biolum/images/Latz_p1.jpg

Img Src: http://www.biolum.org/

slide6

How GFP is being used

  • Tag Cells (to detect specific cells)
  • Act as a reporter gene
    • - link it to another gene to show if it is expressed
  • Expressed in entire animals

Img Src: http://www.bio.umass.edu/microscopy/images/gfp.jpg

Img Src: http://www.mshri.on.ca/nagy/graphics/GFP%20mice.jpg

Img Src: http://www.antville.org/img/pop/gfp.jpg

Img Src: http://www.computerra.ru/pubimages/73944.jpg

nobel prize 2008
Nobel Prize 2008
  • In 2008, 3 scientists were awarded the 2008 Nobel Prize in Chemistry for their work on GFP
pgfp plasmid
pGFP plasmid
  • The plasmid we’re using in the lab
  • 2 genes of interest:
    • GFP gene
      • Codes for the GFP protein
    • ampR gene
      • Codes for the enzyme b-lactamase
      • b-lactamase destroys the antibiotic ampicillin
selecting for transformed cells
Selecting for Transformed Cells
  • Selection = process to determine which E. coli successfully took in a plasmid
  • Achieved through the use of selectable markers
  • Selectable markers = traits that help identify a cell with the plasmid in it (compared to one without it)
  • In our experiment, the ampR gene will serve as the selectable marker

Images taken without permission from http://www.antibioresistance.be/Gifs_Ant/blue2.gif and http://www.antibioticos.it/images/formule%20chimiche/ampicillin.gif

review question
Review Question…
  • What protein does the ampR gene code for?
    • b-lactamase protein
  • What does this protein do?
    • Digests the antibiotic ampicillin
  • How could the ampR gene serve as a selectable marker?
    • Only cells with the pGFP plasmid will make b-lactamase  are resistant to ampicillin
growing e coli
Growing E. coli
  • Bacteria is grown on LB agar
    • LB agar contains all of the nutrients and amino acids E. coli need to survive
    • Other substances such as antibiotics can also be added to the LB agar.
selection process

grown on plain LB agar

E. coli cells that have gone through the tranformation process

grown on LB agar with ampicillin

Selection Process

All E. coli grow (transformed and untransformed)

Only transformed cells grow

controls
Controls
  • Grow E. coli without plasmid (- DNA) on plain LB agar
    • Make sure E. coli can grow
  • Grow E. coli without plasmid (- DNA) on LB/amp
    • Make sure E. coli aren’t already resistant to ampicillin
transformation efficiency
Transformation Efficiency
  • Quantitative measurement of how well the transformation worked.
  • Measures the number of bacteria transformed by 1 mg of plasmid DNA.

= total # of cells growing

amount of DNA spread on plate

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