GFP Discovery and First Applications
Discovery of GFP protein • 1960: Osamu Shimomura began studying bioluminescence in Aequorea victoria. • 1962: Reported properties of aequorin, a protein which glows blue around Ca 2+ ions • 1974: Established function of GFP and energy transfer between GFP and aequorin • 1978: Reported structure of GFP, setting much research in motion
First Applications of GFP • 1988: Martin Chalfie becomes interested in GFP and expressing it in proteins in C. elegans • 1992: Obtained gene from Prasher (first to sequence GFP gene) and successfully inserted it into E. Coli and C. elegans • 1994: Published paper “GFP as a marker for gene expression”, popularizing the use of GFP for tagging
GFP Function and Modifications Shamii, Natalie, Stacy, Dimitri
Modifications • E-GFP-enhanced version of original gene that incorporates mutations that occurred in other laboratories. • -gives about two orders of magnitude greater sensitivity in human and mammalian cells than the original jellyfish gene • E-BFP, a version of E-GFP that gives off a bluish light. • By using both E-GFP and E-BFP, two different proteins could be marked simultaneously in the same experiment. • Others have extended the colour palette beyond green and blue allowing researchers to give various proteins and cells different colours.
Modification Continued • Superfolder GFP • series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folded peptides • Cycle 3 mutant makes it 42 times more fluorescent than wild-type GFP and reduce hydrophobicity of GFP and suppress aggregation • Created a vector system that delivers the DNA into the right organism or the right cells.
Structure • Beta barrel with a alpha helix going through the center. • -alpha helix contains the chromophore (which makes the protein glow) • -side chains of beta barrel create reactions that produce the chromophore • Made out of 238 amino acids
Roger Tsien • His family are descendants of the royal family of the Kingdom of Wuyue. • Got bachelors degree from Harvard and Ph. D from Cambridge. • Well known for tracking calcium in DNA and proteins. • Revolutionized nerve+ cell biology by allowing people to watch molecules in real time.
HIV & Cancer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses1.html Cancer HIV Virulence http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses18.html http://www.aps.org/publications/apsnews/200705/images/aids_photo_web.jpg
Red Cats • December 17th, 2007 • Professor Kong Il-keung • South Korea • Genetically modified cloned cat • Closer genetically to humans than mice • No variance in genes of clones • Perfect for study of inherited diseases • 250 of feline genetic diseases are found in humans too
Human knee cells enhanced with fluorescent green GFP cells
GFP use • Keung used GFP to show that the cats could be genetically modified • Evidence of modification when cats, when under blue light, glow red • Process • Skin cells taken from Turkish Angora cat • Modified with fluorescent protein from red coral • Nuclear material placed in emptied nucleus of fertilized ovum • Implanted in womb of donor
Normal lighting Blue light
PIGS! • Randy Prather • Professor reproductive biotechnology • University of Missouri, Columbia • Leading scientist in creation of transgenic (inserted genetic material into genome) medicine • Transgenic YFP • Use of YFP (mutant GFP) as yellow marker for modified pig • “These animals prove that we can make genetic modifications to express desired traits…it's possible to change the genetic makeup of the cells to prevent the body's rejection of transplanted organs”
Malaria • FIGHT! • No easy way to differentiate male and female mosquito larvae • Use GFP to make male testes glow • Sterilize males and release only them into environment • Females mate with them and yield no fruit (ie baby mosquits) • Therefore you can eradicate a malaria mosquito population
Neural Growth • So…there’s a momma mouse and a dadda mouse… • One has GFP neurons • Other has BFP (blue) neurons • They make babies • You get a baby with blue and green neurons…YAY! • Tells how mother and father’s genes are networked in the • Can watch how the vie for space on neural muscle tissues
Brainbow • The brainbow is the coolest of all of the GFP applications • Brainbow is a combination of brain and rainbow • The brain rainbow, or brainbow, has been used to color neural circuits
Brainbow CONTINUED • This neural coloring helps scientists to map the neural circuits • They combine yellow, green and cyan fluorescent proteins to create a mix of ninety different hues and colors!