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
Overall Goal of Lab Experiment
• Use genetic engineering techniques to insert
the GFP gene into E. coli
Plasmid containing
gene of interest
Protein we want to
produce
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
Structure of the GFP Protein
Img Src: http://wwwchem.leidenuniv.nl/metprot/armand/images/029l.jpg
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/
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%20mic
e.jpg
Img Src: http://www.antville.org/img/pop/gfp.jpg
Img Src: http://www.computerra.ru/pubimages/73944.jpg
Nobel Prize 2008
• In 2008, 3 scientists were awarded the 2008
Nobel Prize in Chemistry for their work on
GFP
pGLO plasmid
• The plasmid we’re using in the lab
• 3 genes of interest:
– GFP gene
• Codes for the GFP protein
– Bla gene
• Codes for the enzyme b-lactamase
• b-lactamase destroys the antibiotic
ampicillin
– araC gene
• Codes for the araC protein
Arabinose operon
• The arabinose operon in bacteria consists of
the following:
Usually, the araC protein
binds to the arabinose
operon operator 
prevents transcription
When arabinose is
present, it binds to the
araC protein -> can’t bind
to operator  RNA
polymerase can continue
Modified arabinose operon
• Scientists modified the arabinose operon in
the pGLO plasmid to express the GFP gene.
araC protein binds to
the operator 
prevents transcription
When arabinose binds to
araC it can no longer
bind to operator  GFP
gene is transcribed and
translated
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 bla 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…
• What protein does the bla gene code for?
– b-lactamase protein
• What does this protein do?
– Digests the antibiotic ampicillin
• How could the bla gene serve as a
selectable marker?
– Only cells with the pGLO plasmid will make blactamase  are resistant to ampicillin
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
All E. coli grow
(transformed and
untransformed)
Only
transformed
cells grow
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
• Grow E. coli with plasmid (+ DNA) on
LB/amp
– Make sure transformation didn’t kill E. coli
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