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

开发基于细胞的蛋白质组库的荧光条形码系统

• 批准号: 1066806
• 负责人: David Colby
• 金额: $ 28.16万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1066806&HistoricalAwards=false
• 关键词: Development; fluorescent; bar; coding; system

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.

知识优势：对蛋白质组的分析使我们能够前所未有地深入了解细胞功能背后的动态分子网络。这里提出的计划将开发一个强大的新工具，大大加快蛋白质组学研究的步伐。基于细胞的蛋白质组文库，其中单个蛋白质与报告蛋白（如GFP）融合，敲除或过度表达，已成为量化蛋白质组和识别参与特定细胞功能的蛋白质的宝贵手段。然而，从这些文库中收集数据受到用于识别成员的方法的限制，包括对DNA条形码进行测序或单独测试克隆。本计划将发展一套荧光条形码系统，以进行单细胞分析，并利用多色流式细胞术快速鉴定文库成员。当与感兴趣的属性的荧光读数相结合时，这将是一个特别强大的工具。为了实现这一目标，一个蛋白质家族将被设计用于细胞表面表达，它将唯一地识别每个文库成员，就像细胞表面标记被用来识别血液中无数的免疫细胞一样。蛋白Titin的片段将被用作支架来呈现表位标签的组合；每个表位标签将对应条形码的一种颜色，而每个表位标签的拷贝数将定义颜色？年代的强度。然后将对该系统进行表征，包括确定细胞表面Titin片段的表达对正常细胞生理的影响。最后，新系统将在6029个成员gfp融合蛋白质组酵母文库中实施和测试，通过流式细胞术实现对所有6029个蛋白质的单细胞测量，采样速度为每分钟200倍。更广泛的影响：除了开发一种广泛适用的使能技术外，这项工作的影响还将通过将高中教师带入实验室参与研究并开发相关的生物工程课程模块与学生分享而扩大。高中教师的研究经历也使学生受益，因为它增强了教师对科学研究的理解，使他们跟上科学发展的步伐；这些知识对他们培养有抱负的工程师和科学家的能力至关重要。接触现代生物学；这是高度量化的；对高中科学教师来说是至关重要的，他们中的许多人可能仍然把生物学作为一门描述性科学来教授。科学教师将从服务于城市和少数民族学生比例较高的郊区的地区公立学校招聘。