GFP: Lighting Up Life You can observe a lot by watching. Yogi Berra - - PowerPoint PPT Presentation

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GFP: Lighting Up Life You can observe a lot by watching. Yogi Berra - - PowerPoint PPT Presentation

Sep 17, 2023 27 likes •392 views

GFP: Lighting Up Life You can observe a lot by watching. Yogi Berra My companions and I then witnessed a curious spectacle. . .The Nautilus floated in the midst of. . . truly living light. . . an infinite agglomeration of colored. . . globules

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My companions and I then witnessed a curious spectacle. . .The Nautilus floated in the midst of. . . truly living light. . . an infinite agglomeration of

  • colored. . . globules of diaphanous jelly. . .

Twenty Thousand Leagues Under the Sea – Jules Verne Now it is such a bizarrely improbable coincidence that anything so mind- bogglingly useful could have evolved purely by chance that some thinkers have chosen to see it as a final and clinching proof of the nonexistence of God. The Hitchhiker’s Guide to the Galaxy – Douglas Adams

GFP: Lighting Up Life

You can observe a lot by watching. Yogi Berra

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Wilhelm Röntgen Santiago Ramón y Cajal Camillo Golgi

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Ultramicroscope Richard Zsigmondy Chemistry, 1925 Phase Contrast Microscope Frits Zernike Physics, 1953 Large- Array Radio Telescopes Martin Ryle Physics, 1974 Electron Microscope Ernst Ruska Physics, 1986 X-ray Crystallography Physics, 1915 Lawrence Bragg William Bragg Nuclear Magnetic Resonance Physics, 1952

  • E. M.

Purcell Felix Bloch Scanning Tunneling Microscope Physics, 1986 Heinrich Rohrer Gerd Binnig Computer Assisted Tomography Physiology or Medicine, 1979 Godfrey Hounsfield Allan Cormack Magnetic Resonance Imaging Physiology or Medicine, 2003 Peter Mansfield Paul Lauterbur

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Sydney Brenner José Zadunaisky Bob Perlman

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Sydney Brenner Bob Horvitz John Sulston Caenorhabditis elegans

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β-galactosidase Activity mec-9 Antibody Staining MEC-7

Hongping Du

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Aequorin + Ca++ + GFP light Aequorin + Ca++ light

Paul Brehm Osamu Shimomura Aequorea victoria

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Douglas Prasher

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The GFP Fluorophore

Tyr66 Phe64 Ser65

. . . . . . NH-

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1 TACACACGAA TAAAAGATAA CAAAGATGAG TAAAGGAGAA GAACTTTTCA CTGGAGTTGT 61 CCCAATTCTT GTTGAATTAG ATGGTGATGT TAATGGGCAC AAATTTTCTG TCAGTGGAGA 121 GGGTGAAGGT GATGCAACAT ACGGAAAACT TACCCTTAAA TTTATTTGCA CTACTGGAAA 181 ACTACCTGTT CCATGGCCAA CACTTGTCAC TACTTTCTCT TATGGTGTTC AATGCTTTTC 241 AAGATACCCA GATCATATGA AACAGCATGA CTTTTTCAAG AGTGCCATGC CCGAAGGTTA 361 CAAGTTTGAA GGTGATACCC TTGTTAATAG AATCGAGTTA AAAGGTATTG ATTTTAAAGA 421 AGATGGAAAC ATTCTTGGAC ACAAATTGGA ATACAACTAT AACTCACACA ATGTATACAT 481 CATGGCAGAC AAACAAAAGA ATGGAATCAA AGTTAACTTC AAAATTAGAC ACAACATTGA 541 AGATGGAAGC GTTCAACTAG CAGACCATTA TCAACAAAAT ACTCCAATTG GCGATGGCCC 601 TGTCCTTTTA CCAGACAACC ATTACCTGTC CACACAATCT GCCCTTTCGA AAGATCCCAA 661 CGAAAAGAGA GACCACATGG TCCTTCTTGA GTTTGTAACA GCTGCTGGGA TTACACATGG 721 CATGGATGAA CTATACAAAT AAATGTCCAG ACTTCCAATT GACACTAAAG TGTCCGAACA 781 ATTACTAAAA TCTCAGGGTT CCTGGTTAAA TTCAGGCTGA GATATTATTT ATATATTTAT 841 AGATTCATTA AAATTGTATG AATAATTTAT TGATGTTATT GATAGAGGTT ATTTTCTTAT

λGFP10

EcoRI EcoRI

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Ghia Euskirchen

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Yuan Tu

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Bill Ward

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Green Fluorescent Protein: A New Marker for Gene Expression The Aequorea victoria Green Fluorescent Protein Needs No Exogenously-Added Component to Produce a Fluorescent Product in Prokaryotic and Eukaryotic Cells Martin Chalfie, Yuan Tu, Ghia Euskirchen, William W. Ward, Douglas C. Prasher Science 263: 802-805, 1994 Green Fluorescent Protein as a Marker for Gene Expression

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Tulle Hazelrigg Sarah Chalfie

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Shengxian Wang and Tulle Hazelrigg

Shengxian Wang

Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis Nature 369: 400-403, 1994

Tulle Hazelrigg

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Advantages of GFP as a Biological Marker 1. Heritable 2. Relatively Non-invasive 3. Small and Monomeric 4. Visible in Living Tissues

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Improving GFP

Tyr66 Emission Ser65 Excitation

Roger Tsien

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Papers Using Green Fluorescent Protein

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The First Human GFP Transgenic?

Ang Lee

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PVM AVM PLM ALM Pmec-17 gfp

Gene Expression

Yun Zhang

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FLP FLP FLP

EGL-44::YFP EGL-46::CFP MERGED

Co-expression

Ji Wu

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Pmec-4 cfp mec-4::yfp

Dattananda Chelur

Protein Localization

MEC-1 MEC-9 MEC-5 MEC-7 MEC-12 MEC-4 MEC-10 MEC-6 MEC-2

β-tubulin α-tubulin collagen Kunitz/EGF proteins DEG/ENaC PHB-domain proteins paraoxonase

UNC-24

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unc-51 mec-7 Wild Type

Mutant Screens and Characterizstion

Hongping Du

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gfp rab-3

Mike Nonet

Visualizing Synapses

wild type mec-15

Alex Bounoutas Pmec-7

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Yun Zhang

Cell Isolation

Irini Topalidou

Touch neurons FLP neurons

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The Problem with C. elegans Electrophysiology

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Bob O’Hagan

Cell-specific Electrophysiology

MEC-4 MEC-10 MEC-6 MEC-2

Miriam Goodman

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Interaction of Leucine Zippers Reconstitution of Functional GFP recGFP NZGFP

NZ CZ linker NGFP CGFP linker

CZGFP

From Promoter A From Promoter B

A B

Non-covalent Reconstitution of GFP

Lynne Regan Indraneel Ghosh, Andrew D. Hamilton, and Lynne Regan (2000) Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. J. Am. Chem. Soc. 122: 5658–5659.

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Refining Cell Labeling

mec-3 egl-44 FLP Intestine Pmec-3 nzgfp & Pegl-44 czgfp PVD PLM AVM ALM PVM Hypodermis HSN Other neurons Shifang Zhang Chuck Ma

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L1 L4 Adult Control Colchicine

Mutagenesis Colchicine

Alex Bounoutas Chuck Ma Leslie Emtage praja gfp Pmec-18 MEC-3 MEC-7 + MEC-12 microtubules

Homeodomain Transcription Factor β-tubulin α-tubulin

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Madeline Friedlen Vivian Chalfie Eli Chalfie

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Postdoctoral Scientists Anahita Amiri Guy Caldwell Lucinda Carnell Anne Chambers Dattananda Chelur Brian Coblitz Monica Driscoll Chip Ferguson Nikolaos George Miriam Goodman Jang Hee Hahn Chris Li Charles Ma Chris Martin Shohei Mitani Erich Schwarz Anne Toker Irini Topalidou Millet Treinin Bill Walthall Jeff Way Eve Wolinsky Graduate Students Alex Bounoutas Andrea Calixto Xiaoyin Chen Yushu Chen Hongping Du Anne Duggan Lesley Emtage Glen Ernstrom Ghia Euskirchen Jaime García- Añoveros Guoqiang Gu Mingxia Huang Siavash Karimzadegan Charles Keller John Kratz Joy Liang Shujun Luo Cathy Savage Jim Taub Ji Wu Yi-Chun Wu Ding Xue Shifang Zhang Yun Zhang Undergraduates Macy Au Paul Auwaeter Peggy Brickman Kate Brauman John Byun Alex Chang Victor Chang Iris Chin Bradley Collins Michael Dreyfuss Angela Georgopoulos Rachel Goldstein Judith Green Peter Homer Rafaz Hoque James Hudspeth Shari Jawetz Paul Josephson Eric Kanter Lexy Kovach Joe Lau Sam Lee Lorraine Lothringer David Meshoulam Jeremy Mindich Mariya Rosenblit Julie Rosenthal Aaron Scheffler Shai Shaham Jay Srinivasan Leslie Vosshall Molly Weiner Chris William Technicians Margaret Barnes Elke Bergholz Lei Chen Ellen Dean Nora Hom Maud Kinnell Connie Mauoka Evelyn Reilly Yuan Tu Yingzi Xue Andrea Yao Faculty Visitors Christian Neri Jonathan Rothblatt