RFP 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
Overall Goal of Lab Experiment • Use genetic engineering techniques to insert the mfp gene into E. coli Plasmid containing gene of interest Protein 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
Structure of RFP (protein) Img Src: http://web.aibn.uq.edu.au/cbn/research_biomolecular.htm
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 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.conncoll.edu/ccacad/zimmer/GFP-ww/lukyanov.html Img Src: http://www.mshri.on.ca/nagy/graphics/GFP%20mice.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.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 b-lactamase • b-lactamase destroys the antibiotic ampicillin • araC gene • Codes for the araC protein
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 Arabinose operon • The arabinose operon in bacteria consists of the following:
When arabinose binds to araC it can no longer prevent transcription mfp gene is transcribed and translated Modified arabinose operon • Scientists modified the arabinose operon in the pARA-R plasmid to express mfp gene.
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? • 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 pARA-R plasmid will make b-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.
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 • 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