CAREER: A novel approach for deciphering cellular metabolic phenotypes using tandem mass spectrometry

这项建议的总体研究目标是更好地了解细胞新陈代谢，以便有目的地操纵细胞行为。新陈代谢是活细胞将营养物质转化为产品的基本过程。目前关于代谢在细胞行为中的作用的知识状态是有限的，因为很难精确和准确地测量细胞内的代谢通量。该项目将使用一种新的测量技术--串联质谱仪，以更高的精确度和准确度量化细胞的代谢状态，从而有助于了解微生物代谢和细胞生理学。拟议的工作将通过以下方式增加对科学的理解：(1)引入一种新的代谢通量分析(MFA)方法；(2)提供验证通量结果和识别潜在误差来源的改进技术；(3)开发用于MFA的新算法和易于使用的工具；以及(4)提高对两种重要模式微生物--大肠杆菌和酿酒酵母--代谢网络运行的了解。这项提议的教育目标是促进定量系统生物学原理的教学，作为化学工程教育的一个组成部分，并加强代表不足的群体参与生物研究。具体地说，教育方法是：(1)向当地首屈一指的HBCU特拉华州立大学的少数族裔本科生提供暑期研究机会；(2)开发新的课程材料，并在特拉华大学开发关于生物蛋白质生产、回收和纯化的激动人心的Capstone实验室课程。该项目还为跨学科环境下的学生教育和培训提供了独特的机会。学生将在微生物细胞培养、代谢工程、代谢组学、通量分析、质谱学和定量建模方面获得经验。这些经验将使学生站在代谢工程和新兴的定量系统生物技术领域的前沿。

The overall research objective of this proposal is to develop better understanding of cellular metabolism to allow purposeful manipulation of cell behavior. Metabolism is the fundamental process by which living cells convert nutrients into products. The current state-of-knowledge regarding the role of metabolism in cellular behavior is limited due to difficulties in measuring intracellular metabolic fluxes precisely and accurately. This project will contribute to the knowledge of microbial metabolism and cell physiology by quantifying the metabolic state of cells at a much higher level of precision and accuracy using a novel measurement technique, tandem mass spectrometry. The proposed work will add to the scientific understanding by: (1) introducing a new method of metabolic flux analysis (MFA); (2) providing improved techniques for validating flux results and identifying potential sources of errors; (3) developing new algorithms and easy to use tools for MFA; and (4) improving knowledge of metabolic network operation for two important model microbes, Escherichia coli and Saccharomyces cerevisiae.The educational objective of this proposal is to promote teaching of quantitative systems biology principles as an integral part of chemical engineering education and to enhance the participation of underrepresented groups in biological research. Specifically, the educational approach is to: (1) provide summer research opportunities to minority undergraduate students from Delaware State University, a local premier HBCU; and (2) develop new course material and a stimulating capstone laboratory course on biological protein production, recovery and purification at the University of Delaware. This project also provides unique opportunities for student education and training in an interdisciplinary environment. Students will gain experience in microbial cell culture, metabolic engineering, metabolomics, flux analysis, mass spectrometry and quantitative modeling. These experiences will place the students at the forefront of metabolic engineering and the emerging field of quantitative systems biotechnology.