DETECTION Molly Petrous Mackenzie Reetz Madison Salomon - PowerPoint PPT Presentation

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

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