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RFP Transformation LabRFP Transformation Lab
Images taken without permission from http://web.aibn.uq.edu.au/cbn/research_biomolecular.htm , http://bioinfo.biotec.or.th/Picture/Cell%20Tutorial/image005.jpg, http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Jellyfish.jpeg
• Use genetic engineering techniques to insert the mfp gene into E. coli
Overall Goal of Lab Experiment
Plasmid containing gene of interestProtein we
want to produce
It all started with GFP (Green Fluorescent Protein)
• Naturally produced in Jellyfish– Aequorea victoria
• The first bioluminescent protein discovered in the 1960s
• Glows green 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
RFP (Red Fluorescent Protein)
• Naturally produced in Discosoma sp. (a sea anemone)
• Discovered in 1999
• Similar to GFP
• Led to the discovery of several of GFP-like proteins
Img Src: http://www.conncoll.edu/ccacad/zimmer/GFP-ww/lukyanov.html, http://0.tqn.com/d/saltaquarium/1/0/2/X/redmushroomtom_400.JPG
Why Is Bioluminescence Useful in Nature?
• Attract Mates
• See Food
• Defense
Img Src: http://www.biolum.org/
Img Src: http://www.sio.ucsd.edu/explorations/biolum/images/Latz_p1.jpg
Img Src: http://www.biotech-now.org/health/2011/09/doggonn-it-this-kitty-may-be-researchers%E2%80%99-new-best-friend
Img Src: http://www.conncoll.edu/ccacad/zimmer/GFP-ww/lukyanov.html
Img Src: http://www.mshri.on.ca/nagy/graphics/GFP%20mice.jpg
How RFP and other similar proteins are 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.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
pARA-R plasmid• The plasmid we’re using in the lab• 3 genes of interest:
– mfp gene• Codes for the RFP protein• Modified to glow brighter than original rfp
– ampR gene• Codes for the enzyme -lactamase• -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 pARA-R plasmid to express mfp gene.
When arabinose binds to araC it can no longer
prevent transcription mfp 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 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…
• What protein does the ampR gene code for?– -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 pARA-R plasmid will make
-lactamase 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
E. coli cells that have gone through the tranformation process
grown on plain LB agar
grown on LB agar with ampicillin
All E. coli grow (transformed and untransformed)
Only transformed cells grow