DETECTION Molly Petrous Mackenzie Reetz Madison Salomon - - PowerPoint PPT Presentation

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DETECTION Molly Petrous Mackenzie Reetz Madison Salomon - - PowerPoint PPT Presentation

Mar 22, 2023 469 likes •702 views

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

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SLIDE 1

Meredith Gentry Emma Goldstein Grace Hershberger Sam Higgins Piper Inglis Tyler Knox Molly Petrous Mackenzie Reetz Madison Salomon Amna Sohail Shafer Stellema

  • Mrs. Kelly Bozoian

Instructor

  • Mr. Paul Legge

Consultant

EARLY MELANOMA DETECTION LUMINESCENT BIOSENSOR

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SLIDE 2

Yeast based malignant melanoma biosensor Luminescent cream

  • Cheap
  • Efficient, early diagnosis
  • Readily available

INTENT

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SLIDE 3

Yeast containment Safe strand of yeast Protective gear Safety precautions

SAFETY

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SLIDE 4

MELANOMA RESEARCH

Figure 2 Figure 2 Figure 3

Figure 2 & 3: Early Stage Melanoma ma Sourc rce: e: Natio tional l Cancer cer Institut itute Figure 1: Melanoma ma Mortality y Rate tes in U.S. (2000-2004) Sourc rce: e: Natio tional l Cancer cer Institut itute

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SLIDE 5

MELANOCYTE PATHWAYS RESEARCH

Melanoc

  • cyte Pa

Pathwa ways ys Source rce: : Natio ional l Center ter for Biotechn technolo logy gy Informatio tion

Figure 4 Figure 5

Melanoc

  • cyte Pa

Pathwa ways ys Source: rce: Natur ure Revi Reviews ws Clinica cal l Oncolo logy gy

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SLIDE 6

FGFR-1 & FGF2 RESEARCH

Figure 6 Figure 7

Source: rce: Beatson son Instit itut ute e for r Cancer er Re Resea earc rch Source: rce: Cell Adhesi esion and Matri rix Re Resea earc rch h Center er, , Univer ersi sity ty of Alaba bama

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SLIDE 7

FGFR-1 & FRS2 RESEARCH

Source: rce: Natio ional l Center ter for Biotechn technolo logy gy Informatio tion

Source: ce: Nation ional l Center er for Biotec echnology logy Inform rmation Figure 9 Figure 8

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SLIDE 8

3A General Assembly Dr. Goebl yeast Promega Lab mishaps

PROTOCOL

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SLIDE 9

YEAST BIOSENSOR PHASES & BIOBRICKS

  • 1. Detection of Input (FGF2

Ligand)

  • 2. Activation of Signaling

Pathway

  • 3. Activating the Response

(Output – Luciferase/Renilla Expression)

FGFR-1

FRS2

Luciferase & Renilla YEAST CHASSIS

1 2 3

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SLIDE 10

PHASE ONE: DETECTION OF INPUT

 Fibroblast Growth Factor Receptor-1 (FGFR-1)

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SLIDE 11

PHASE TWO: ACTIVATION OF SIGNALING PATHWAY

 Fibroblast Growth Factor Receptor Substrate 2 (FRS2)

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SLIDE 12
  • BBa_K9

K976009: 009: TDH3 Pro romot

  • ter

er + Y Yeast t Kozak + FGFR-1/FRS2 1/FRS2 Complex +TEF1 Terminat nator

  • r

B C D E

A

  • FGFR-1 & FRS2 Fusion Protein

Translational Unit BioBrick

DETECTION AND ACTIVATION

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SLIDE 13

PHASE THREE: ACTIVATING RESPONSE

 Autonomously Replicating Sequence Binding Factor 1 (ABF1)

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SLIDE 14

PHASE THREE: ACTIVATING RESPONSE

 Promoter Control psbA2 Reference Dual Luciferase Assay

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SLIDE 15

A B C D E F G H

  • 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

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SLIDE 16

C D

A

B

  • Bba_K976008: Yeast ADH1 Promoter + Firefly Luciferase + ADH1 Promoter +

Renilla Luciferase

PHASE THREE: ACTIVATING RESPONSE

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SLIDE 17

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

PHASE THREE: ACTIVATING RESPONSE

 Yeast ADH1 promoter and pKC3161 Yeast Plasmid Expression Vector

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SLIDE 18

BBa_K976008 failed to luminesce Developed new plasmid and expression vector

RESULTS

Dual Luciferase System Growth

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SLIDE 19

Short term:

  • BBa_K976008

luminesce

  • Synthesize & transform

FGFR-1 & FRS2 BioBrick

Long term:

  • Signaling pathway phase
  • Test FGFR-1 and FGF2

binding

  • Cream development

NEXT STEPS

FGFR-1 & FGF2 binding Source: rce: Instit itut ut Européen péen de Chimie e et et Biologi gie e (IECB)

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SLIDE 20

SPONSORS

Bill Vincent

  • Dr. Goebl & Dr. Chun
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SLIDE 21

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 Information. 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.>