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St. Dominic SMART Team: Elana Baltrusaitis, Allyson Bigelow, Rachel Brielmaier, Pamela Burbach, Jake Dowler, Johnny Fuller, Caroline Hildebrand, Teagan Jessup, Molly Jordan, Josh Kramer, Jenna Lieungh, Alex Mikhailov, Pat O’Grady, Andrew Pelto, Quin Rowen, Rachelle Schmude, Bobby Schultz, Katherine Seubert, Alex Sherman, Parker Sniatynski, Alex Venuti, Erin Verdeyen, and Molly Wetzel Teacher Advisor Donna LaFlamme Mentors: Nathan Duncan and Françoise Van den Bergh, Ph.D., Medical College of Wisconsin Luciferase is the generic name for an enzyme responsible for bioluminescence reactions and is commonly associated with fireflies. It is also found in many other organisms including bacteria, fungi, anemones, and dinoflagellates. Since the gene for the North American firefly (Photinus pyralis) luciferase was cloned in 1985, scientists have been genetically engineering the gene into living cells. The luciferase reaction is now widely used in scientific research to study protein production in cells, to analyze gene promoter activity, to study stem cell function in vivo, and in cancer studies, to trace the metastasis of cancer cells in living test animals. The scientific study of the luciferase enzymes themselves is also continuing. In recent research, single amino acid mutations to the active site cause the emission of different colored light in a predictable way. The uses of and improvements in bioluminescent imaging are increasing exponentially in cell biology, molecular biology, and in medical research. LUCIFERASE FACTS TO KNOW LUCIFERASE IN RESEARCH • Luciferase is an enzyme found in the lanterns of fireflies in cells called photocytes. • Its function is to catalyze a bioluminescence reaction in which the substrate luciferin is oxidized to oxyluciferin. • Bioluminescence is the production of light by a chemical reaction within living organisms. • Fireflies use bioluminescence for communicating, attracting mates, luring in prey, and self-defense. Firefly Luciferase (Luciola cruciata) Physical Model based on PDB File: 2D1R [1] Fat Cell Growth Over 12 Weeks ACTIVE SITE OF FIREFLY LUCIFERASE (Luciola cruciata) Japanese Firefly (Luciola cruciata) Scientists discovered a fat progenitor cell with the help of luciferase. The progenitor cells were injected into mice genetically unable to produce fat. The luciferase gene turned on in mature fat cells and allowed the researchers to track the development of the fat cells over time in the living animal. [2] http://2.bp.blogspot.com/_VVjUm4hzpvo/SSPbxn1OnJI/AA AAAAAABd8/UnJeVFSM9RI/s400/firefly.jpg IVIS® imaging system which is used to pick up the faint light given off by the oxyluciferin in the cells expressing luciferase. ACTIVE SITE OF WILD TYPE AND MUTANT S286N LUCIFERASE FIREFLY BIOLUMINESCENCE REACTION Ile 288 DLSA RasMol Image based on PDB File: 2D1R [1] Step 1: Luciferin and magnesium-ATP react to form the luciferyl-AMP intermediate. Step 2: Luciferin-AMP reacts with O2 to form excited oxyluciferin which emits a photon of light as it returns to its ground energy state. 1885- Raphael DuBois ground up beetle lanterns in hot and cold water obtaining no luminescence in the hot water. He named luciferin and luciferase. Hot water destroyed luciferase. [3] 1955-Luciferase was purified as a protein by Green and McElroy The luciferase active site provides a hydrophobic pocket for producing the electronically unstable oxyluciferin. Single amino acid mutations of Ile 288 to Val288 and to Ala288 and of Ser 286 to Asn 286 results in the emission of lower energy light. [1] See below. Wild Type(yellow) I288V (orange) l288A (reddish-pink) S286A(red) 1947- W.D. McElroy discovered that ATP is an essential requirement for the bioluminescence reaction and light production is proportional to ATP. [3] 1959- McElroy and Seliger discovered the quantum yield of the oxidation of firefly luciferin to be 90% efficient.[3] bioluminescencehttp://upload.wikimedia.org/wikipedia/en/2/27/William_David_McElroy.jpg http://upload.wikimedia.org/wikipedia/commons/f/f7/Luciferase.png [1]Primary Citation : T. Nakatsu, et. al. Structural basis for the spectral difference in luciferase bioluminescence. Nature, 2006, 440, 372-376 1976- DeLucca shows that the firefly bioluminescence reaction is a two step process requiring ATP, O2, and luciferin. 1967- Determination of picogram amounts of ATP using luciferin-luciferase enzyme system by G.E. Lyman and J.P. DeVincenzo. [4] [2] M. S. Rodeheffer et. al. Identification of White Adipocyte Progenitor Cells In Vivo. Cell, 2008, 135240-249 [3] H. Fraga, Firefly luminescence: A historical perspective and recent developments. Photochemical and Photobiological Sciences, 2008, 7, 146-158 [4] J. Lee, Bioluminescence Timelines, University of Georgia. A SMART Team project supported by the National Institutes of Health (NIH)-National Center for Research Resources Science Education Partnership Award (NCCR-SEPA). Wild-Type DLSA S286N Mutant Ile 288 is being rotated so it is not packing the intermediate analogue, DSLA ,tightly in the mutant active site. This changes the color of the emitted light from green to lower energy red light because excited oxyluciferin is allowed to lose energy before it emits light. [1] [1] 1961-The structure and synthesis of luciferin was determined by White, Field, McElroy, and McCapra.[4] Ile 288 1985-Marlene DeLucca and colleagues cloned the gene for firefly luciferase and expressed the gene in E. coli bacteria. 1990/91-Mutant luciferases discovered that produce different colors of light http://hongkiat.s3.amazonaws.com/colorfulwp/Rainbow_Ocean__by_Thelma1.j pg Present- luciferase/luciferin is used to locate tumors and monitor the spread of cancer in test animals 1996- E. Conti, N. Frank, and P. Brick crystallized firefly luciferase and obtained an x-ray structure at 2 angstroms resolution. 2010-St.Dominic SMART Team designs physical model of firefly luciferase (2d1r)