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Abstract/Syllabus:
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Howarth, Mark, and Anthony Leung, 7.341 Brightening up Life: Harnessing the Power of Fluorescence Imaging to Observe Biology in Action, Fall 2006. (Massachusetts Institute of Technology: MIT OpenCourseWare), http://ocw.mit.edu (Accessed 07 Jul, 2010). License: Creative Commons BY-NC-SA
Ribbon diagram of the crystal structure of green fluorescent protein. (Image courtesy of RCSB Protein Data Bank entry 1EMA.)
Course Highlights
This course features readings and assignments.
Syllabus
Summary
One summer in the 1960s a young Japanese researcher, with the help of a few high school students, chopped up ten thousand jellyfish. As a by-product of this harvest, they isolated a green fluorescent protein (GFP). Since then, GFP has triggered a revolution in our understanding of gene expression and signaling in live cells. In this seminar, we will examine how this small protein generates fluorescence, i.e. absorbs light of one wavelength and emits light of a longer wavelength. We will discuss how the color palette has been extended from green to blue, red and many other colors, based on protein engineering of GFP and the study of vividly colorful coral reefs. We will then investigate how these fluorescent proteins can be used to track the motion of DNA, RNA and protein in living cells, as well as to see waves of signaling molecules propagate across a cell. GFP is also a powerful tool for fluorescent imaging of whole organisms, from worms to mice, and we will see how it has been used in tracking the spread of cancer cells, controlling malaria and in understanding how neuronal connections form. In this seminar, we will explore this wonderful protein as well as other important methods and reagents for fluorescent imaging.
Course Format
An essential tool in science, as in life, is learning how to evaluate evidence to come to a conclusion. In biology evidence can be complex and can appear to be contradictory. This course is designed to familiarize you with the primary scientific literature, where data are presented, so that you can decide for yourself whether other people's conclusions are well-founded, uncertain, or wrong. The class should be highly interactive. You will be encouraged to express your opinions. Each week we will discuss two primary research papers. We will first consider the objective of the paper and the methods whereby conclusions were reached. We will look carefully at the data and examine whether the authors' conclusions are compelling.
Attendance
The course is based on discussions and contributions in the class. Therefore attendance is essential.
Grading and Assignments
Grading is pass/fail. Attending all classes and completing all assignments satisfactorily will result in a pass.
There will be two assignments in this class:
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Written Assignment: You are required to write a title and an abstract for a manuscript on fluorescent imaging that we will provide. An abstract should include a basic introduction of the subject, the important observations and the implication of the results. The total length of the abstract should be less than 150 words. The manuscript will be distributed on Ses #2, due by Ses #5 and discussed on Ses #6.
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Oral Assignment: You will give a presentation to the class about a paper of your choice. These presentations will be during Ses #10. You should have selected your paper for approval by the instructors by Ses #8. See the lecture notes section (Ses #10) for details about the format.
Calendar
Course calendar.
SES # |
TOPICS |
ASSIGNMENTS |
1 |
General Introduction |
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2 |
Discovery of Green Fluorescent Protein |
Written out |
3 |
Fluorescent Protein Engineering |
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4 |
Photoconversion of Fluorescent Protein Variants to Probe Cellular Dynamics |
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5 |
Labeling Probes Other than Fluorescent Proteins |
Written due |
6 |
Visit to a Fluorescent Microscopy Facility |
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7 |
Visualizing the Central Dogma of Molecular Biology |
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8 |
Fluorescent Sensors of Cell Signaling: FRET |
Oral out |
9 |
Quantitative and Ultra-Sensitive Fluorescent Imaging |
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10 |
Student Presentations |
Oral due |
11 |
Power of Fluorescent Imaging for High-throughput Analysis |
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12 |
Fluorescent Imaging in Living Organisms |
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Course Description
One summer in the 1960s a young Japanese researcher, with the help of a few high school students, chopped up ten thousand jellyfish. As a by-product of this harvest, they isolated a green fluorescent protein (GFP). Since then, GFP has triggered a revolution in our understanding of gene expression and signaling in live cells. In this seminar, we will examine how this small protein generates fluorescence, i.e. absorbs light of one wavelength and emits light of a longer wavelength. We will discuss how the color palette has been extended from green to blue, red and many other colors, based on protein engineering of GFP and the study of vividly colorful coral reefs. We will then investigate how these fluorescent proteins can be used to track the motion of DNA, RNA and protein in living cells, as well as to see waves of signaling molecules propagate across a cell. GFP is also a powerful tool for fluorescent imaging of whole organisms, from worms to mice, and we will see how it has been used in tracking the spread of cancer cells, controlling malaria and in understanding how neuronal connections form. In this seminar, we will explore this wonderful protein as well as other important methods and reagents for fluorescent imaging.
This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.
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Further Reading:
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Readings
Course readings.
SES # |
TOPICS |
READINGS |
1 |
General Introduction |
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2 |
Discovery of Green Fluorescent Protein |
Morise, H., O. Shimomura, F. H. Johnson, and J. Winant. "Intermolecular Energy Transfer in the Bioluminescent System of Aequorea." Biochemistry 13, no. 12 (June 4, 1974): 2656-62.
Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher. "Green Fluorescent Protein as a Marker for Gene Expression." Science 263, no. 5148 (February 11, 1994): 802-5.
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3 |
Fluorescent Protein Engineering |
Yang, F., L. G. Moss, and G. N. Phillips, Jr. "The Molecular Structure of Green Fluorescent Protein." Nat Biotechnol 14, no. 10 (October 1996): 1246-51.
Campbell, R. E., O. Tour, A. E. Palmer, P. A. Steinbach, G. S. Baird, D. A. Zacharias, and R. Y. Tsien. "A Monomeric Red Fluorescent Protein." Proc Natl Acad Sci U.S.A. 99, no. 12 (June 11, 2002): 7877-82.
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4 |
Photoconversion of Fluorescent Proteins Variants to Probe Cellular Dynamics |
Terskikh, A., A. Fradkov, G. Ermakova, A. Zaraisky, P. Tan, A. V. Kajava, X. Zhao, S. Lukyanov, M. Matz, S. Kim, I. Weissman, and P. Siebert. "Fluorescent Timer: Protein that Changes Color with Time." Science 290, no. 5496 (November 24, 2000): 1585-8.
Patterson, G. H., and J. Lippincott-Schwartz. "A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells." Science 297, no. 5588 (September 13, 2002): 1873-7.
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5 |
Labeling Probes Other than Fluorescent Proteins |
Griffin, B. A., S. R. Adams, and R. Y. Tsien. "Specific Covalent Labeling of Recombinant Protein Molecules Inside live Cells." Science 281, no. 5374 (July 10, 1998): 269-72.
Juillerat, A., C. Heinis, I. Sielaff, J. Barnikow, H. Jaccard, B. Kunz, A. Terskikh, and K. Johnsson. "Engineering Substrate Specificity of O6-Alkylguanine-DNA Alkyltransferase for Specific Protein Labeling in Living Cells." Chembiochem 6, no. 7 (July 2005): 1263-9.
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6 |
Visit to a Fluorescence Microscopy Facility |
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7 |
Visualizing the Central Dogma of Molecular Biology |
Janicki, S. M., T. Tsukamoto, S. E. Salghetti, W. P. Tansey, R. Sachidanandam, K. V. Prasanth, T. Ried, Y. Shav-Tal, E. Bertrand, R. H. Singer, and D. L. Spector. "From Silencing to Gene Expression: Real-Time Analysis in Single Cells." Cell 116, no. 5 (March 5, 2004): 683-98.
Bratu, D. P., B. J. Cha, M. M. Mhlanga, F. R. Kramer, and S. Tyagi. "Visualizing the Distribution and Transport of mRNAs in Living Cells." U.S.A. Proc Natl Acad Sci 100, no. 23 (November 11, 2003): 13308-13.
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8 |
Fluorescent Sensors of Cell Signaling: FRET |
Miyawaki, A., J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien. "Fluorescent Indicators for Ca2+ based on Green Fluorescent Proteins and Calmodulin." Nature 388, no. 6645 (August 28, 1997): 882-7.
Reiff, D. F., A. Ihring, G. Guerrero, E. Y. Isacoff, M. Joesch, J. Nakai, and A. Borst. "In Vivo Performance of Genetically Encoded Indicators of Neural Activity in Flies." J Neurosci 25, no. 19 (May 11, 2005): 4766-78.
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9 |
Quantitative and Ultra-Sensitive Fluorescent Imaging |
Phair, R. D., and T. Misteli. "High Mobility of Proteins in the Mammalian Cell Nucleus." Nature 404, no. 6778 (April 6, 2000): 604-9.
Tardin, C., L. Cognet, C. Bats, B. Lounis, and D. Choquet. "Direct Imaging of Lateral Movements of AMPA Receptors inside Synapses." EMBO J 22, no. 18 (September 15, 2003): 4656-65.
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10 |
Student Presentations |
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11 |
Power of Fluorescent Imaging for High-throughput Analysis |
Wheeler, D. B., S. N. Bailey, D. A. Guertin, A. E. Carpenter, C. O. Higgins, and D. M. Sabatini. "RNAi Living-Cell Microarrays for Loss-Of-Function Screens in Drosophila Melanogaster Cells." Nat Methods 1, no. 2 (November 2004): 127-32. Epub (October 21, 2004).
Neumann, B., M. Held, U. Liebel, H. Erfle, P. Rogers, R. Pepperkok, and J. Ellenberg. "High-Throughput RNAi Screening by Time-Lapse Imaging of Live Human Cells." Nat Methods 3, no. 5 (May 2006): 385-90.
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12 |
Fluorescent Imaging in Living Organisms |
Miller, M. J., S.H. Wei, I. Parker, and M. D. Cahalan. "Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node." Science 296, no. 5574 (June 7, 2002): 1869-73.
Amino, R., S. Thiberge, B. Martin, S. Celli, S. Shorte, F. Frischknecht, and R. Menard. "Quantitative Imaging of Plasmodium Transmission from Mosquito to Mammal." Nat Med 12, no. 2 (February 2006): 220-4.
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