Meredith Gentry Emma Goldstein EARLY MELANOMA Grace Hershberger Sam Higgins Piper Inglis Tyler Knox DETECTION Molly Petrous Mackenzie Reetz Madison Salomon LUMINESCENT Amna Sohail Shafer Stellema Mrs. Kelly Bozoian BIOSENSOR Instructor Mr. Paul Legge Consultant
INTENT Yeast based malignant melanoma biosensor Luminescent cream Cheap Efficient, early diagnosis Readily available
SAFETY Yeast containment Safe strand of yeast Protective gear Safety precautions
MELANOMA RESEARCH Figure 2 Figure 2 Figure 3 Figure 1: Melanoma ma Mortality y Rate tes in U.S. (2000-2004) Figure 2 & 3: Early Stage Melanoma ma Sourc rce: e: Natio tional l Cancer cer Institut itute Sourc rce: e: Natio tional l Cancer cer Institut itute
MELANOCYTE PATHWAYS RESEARCH Melanoc ocyte Pa Pathwa ways ys Source: rce: Natur ure Revi Reviews ws Clinica cal l Oncolo logy gy Figure 5 Figure 4 Melanoc ocyte Pa Pathwa ways ys Source rce : : Natio ional l Center ter for Biotechn technolo logy gy Informatio tion
FGFR-1 & FGF2 RESEARCH Source: rce: Beatson son Instit itut ute e for r Cancer er Re Resea earc rch Figure 7 Figure 6 Source: rce: Cell Adhesi esion and Matri rix Re Resea earc rch h Center er, , Univer ersi sity ty of Alaba bama
FGFR-1 & FRS2 RESEARCH Source: ce: Nation ional l Center er for Biotec echnology logy Figure 8 Inform rmation Figure 9 Source: rce: Natio ional l Center ter for Biotechn technolo logy gy Informatio tion
PROTOCOL 3A General Assembly Dr. Goebl yeast Promega Lab mishaps
YEAST BIOSENSOR PHASES & BIOBRICKS FGFR-1 1. Detection of Input (FGF2 Ligand) 1 FRS2 2. Activation of Signaling Pathway 2 3. Activating the Response YEAST CHASSIS Luciferase (Output – Luciferase/Renilla 3 & Renilla Expression)
PHASE ONE: DETECTION OF INPUT Fibroblast Growth Factor Receptor-1 (FGFR-1)
PHASE TWO: ACTIVATION OF SIGNALING PATHWAY Fibroblast Growth Factor Receptor Substrate 2 (FRS2)
DETECTION AND ACTIVATION • BBa_K9 K976009: 009: TDH3 Pro romot oter er + Y Yeast t Kozak + FGFR-1/FRS2 1/FRS2 Complex +TEF1 Terminat nator or A C D E B • FGFR-1 & FRS2 Fusion Protein Translational Unit BioBrick
PHASE THREE: ACTIVATING RESPONSE Autonomously Replicating Sequence Binding Factor 1 (ABF1)
PHASE THREE: ACTIVATING RESPONSE Promoter Control psbA2 Reference Dual Luciferase Assay
PHASE THREE: ACTIVATING RESPONSE A E C D F G H B • Linear Sequence of BBa_K654050 – Promoter Control psbA2 Reference Dual Luciferase Assay BioBrick Key: A & E: psbA2 promoter, 5’UTR, and RBS B: Firefly Luciferase C &G: T1 terminator from E. coli rrnB D & H: TE terminator from coliphage T7 F: Renilla luciferase reporter
PHASE THREE: ACTIVATING RESPONSE A C B D • Bba_K976008: Yeast ADH1 Promoter + Firefly Luciferase + ADH1 Promoter + Renilla Luciferase
PHASE THREE: ACTIVATING RESPONSE Yeast ADH1 promoter and pKC3161 Yeast Plasmid Expression Vector Expres ession ion Vector Credit dit: (Kristin T. Chun, Christopher J. Skalnik, and Mark G. Goebl), Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN
RESULTS BBa_K976008 failed to luminesce Developed new plasmid and Dual Luciferase System Growth expression vector
NEXT STEPS Short term: BBa_K976008 luminesce Synthesize & transform FGFR-1 & FRS2 BioBrick Long term: Signaling pathway phase Test FGFR-1 and FGF2 binding Cream development FGFR-1 & FGF2 binding Source: rce: Instit itut ut Européen péen de Chimie e et et Biologi gie e (IECB)
SPONSORS Bill Vincent Dr. Goebl & Dr. Chun
REFERENCES American Cancer Society "What Are the Key Statistics about Melanoma Skin Cancer?" American Cancer Society . American Cancer Society, 09 Sept. 2012. Web. 28 May 2013. <http://www.cancer.org/cancer/skincancer-melanoma/detailedguide/melanoma-skin-cancer-key- statistics>. Clontech Laboratories, Inc. A Takara Bio Company. Yeast Protocols Handbook. July 2009. Wed. 19 June 2013. <https://mbc-items-production.s3.amazonaws.com/items/documents/007/888/186/original/ADH1%20 Yeast%20Promoter%20PDF.pdf?AWSAccessKeyId=AKIAJE3X3RZJLUZQKLMQ& Expires=1372220771&Signa ture=ezE9tw6Z0vqhkAyHeJdiwlpq4N8%3D&response-content-type=application%2Fpdf>. University of Cambridge’s Team for 2010. International Genetically Engineered Competition. E. glowi. June 2010. Fri. 25 January 2013. <http://2010.igem.org/Team:Cambridge>. Gartside, Michael G. "Loss of Function of Fibroblast Growth Factor Receptor-2: Mutations in Melanoma." Loss of Function of Fibroblast Growth Factor Receptor-2: Mutations in Melanoma (2009): 1-15. American Association for Cancer Research Journals. American Association for Cancer Research, 2009. Web. 25 May 2013. <http://mcr.aacrjournals.org/content/7/1/41.full.pdf>. Kristin T. Chun, Christopher J. Skalnik, and Mark G. Goebl. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN. <mgoebl@iupui.edu>. McNabb, DS. "Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces Cerevisiae." National Center for Biotechnology Informat ion. U.S. National Library of Medicine, 4 Sept. 2005. Web. 19 June 2013. <http://www.ncbi.nlm.nih.gov/pubmed/16151247>. Melanoma Center. "Melanoma - Early Detection." Melanoma Center - Early Detection. N.p., Jan. 2010. Web. 25 May 2013. <http://www.melanomacenter.org/detection/>. Millar, Andrew J. "Luciferase." The Millar Research Group. N.p., n.d. Web. 28 May 2013. <http://millar.bio.ed.ac.uk/lucifer.html>. Sherf, B. A., S. L. Navarro, R.R. Hannah, and K. V. Wood., (1996) Dualluciferase reporter assay: an advanced co- reporter technology integrating firefly and Renilla luciferase assays. Promega Notes 57:2 – 8. [Online.] <http://www.promega.com/pnotes/57/5573a/5573a.html.> Sugiura, Miwa. "Environment of TyrZ in Photosystem II from Thermosynechococcus Elongatus in Which PsbA2 Is the D1 Protein." Journal of Biological Chemistry. Journal of Biological Chemistry, 13 Apr. 2012. Web. 19 June 2013. <http://www.jbc.org/content/287/16/13336.full>. Utah State University’s team for 2011. International Genetically Engineered Machine Competition. Expression Testing and Bioproduct Development. Jun. 2011. Mon. 20 May 2013. <http://2011.igem.org/Team:Utah_State.>
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