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

Img Src: http://web.aibn.uq.edu.au/cbn/research_biomolecular.htm

Structure of RFP (protein)

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

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