Development of a fluorescent bar-coding system for cell-based proteomic libraries

University Of Delaware, Newark DE

Investigators

Abstract

Intellectual Merits: Analysis of the proteome enables unprecedented insights into the dynamic molecular networks that underlie cellular function. The plan proposed here will develop a powerful new tool to greatly accelerate the pace of proteomic research. Cell-based proteomic libraries, in which individual proteins are fused to reporter proteins like GFP, knocked out, or over-expressed, have become an invaluable means of quantifying the proteome and identifying proteins involved in specific cellular functions. However, collecting data from these libraries is limited by the means used to identify members, which include sequencing a DNA barcode or testing clones individually. The proposed project will develop a fluorescent bar- coding system, which would allow for single-cell analysis and rapid identification of library members by means of multi-color flow cytometry. This would be an especially powerful tool when coupled with a fluorescent readout for a property of interest. To accomplish this, a family of proteins will be engineered for cell surface expression that will uniquely identify each library member, much as cell surface markers are used to identify the myriad of immunological cells in blood. Fragments of the protein Titin will be used as a scaffold to present combinations of epitope tags; each epitope tag will correspond to a color of the bar-code, while the number of copies of each epitope tag will define the color?s intensity. The system will then be characterized, including determination of what effect expression of the Titin fragments on the cell surface has on normal cellular physiology. Finally, the new system will be implemented and tested in a 6,029 member GFP-fusion proteomic yeast library, enabling single cell measurement of all 6,029 proteins with 200-fold over sampling per minute, by flow-cytometry. Broader Impacts: In addition to developing a broadly applicable enabling technology, the proposed work's impact will be expanded by bringing high school teachers into the laboratory to participate in research and to develop a related Bioengineering course modules to share with their students. Research experiences for high school teachers also benefit their students, by enhancing the teachers' understanding of scientific research and by keeping them abreast of scientific developments; knowledge essential to their ability to inform aspiring engineers and scientists. Exposure to modern biology; which is highly quantitative; is critical for high school science teachers, many of whom may still teach biology as a descriptive science. Science teachers will be recruited from regional public school serving both urban and suburban areas with a high fraction of minority students.