Metabolism Project

新陈代谢项目

• 批准号: 9073786
• 负责人: JOHN A GERLT
• 金额: $ 42.29万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9073786
• 关键词: Binding Proteins; Core Protein; Enzymatic Biochemistry; Enzymes; Fractionation; Genes; In Vitro; Individual; Knock-out; Lead; Ligands; Maps; Measures; Metabolic; Metabolic Pathway; Metabolism; Microbiology; Modeling; Pathway interactions; Phenotype; Production; Proteins; Research Infrastructure; Structure; Testing; Transcript; Update; base; genome-wide; in vitro activity; in vivo; metabolomics; microbial; novel; novel strategies; overexpression; protein purification; screening; tool; transcriptome sequencing; transcriptomics

PROJECT SUMMARY: METABOLISM PROJECT The Metabolism Project will provide the experimental infrastructure (enzymology, microbiology, transcriptomics, and metabolomics) for testing the EFI's sequence/structure-based tools for predicting in vitro enzymatic activities and in vivo metabolic functions. In using those tools, the Metabolism Project will take the lead in developing integrated strategies for discovering novel metabolic pathways and providing direction for enhancing the tools. The Specific Aims of the Metabolism Project are: 1) coordinate selection of targets for protein production by the Protein Core, protein purification and ligand screening by the Ligand Discovery Project, and integrative pathway mapping by the Modeling Project; 2) measure in vitro enzymatic activities of targets produced by the Protein Core to validate predictions from integrative pathway mapping by the Modeling Project; 3) construct null (knockout) strains of genes encoding enzymes for which in vitro activities are predicted by the Modeling Project; 4) perform targeted transcript analyses by qRT-PCR and, when appropriate, genome-wide transcriptomics by RNA-Seq to confirm predicted metabolic functions predicting by the Modeling Project; 5) analyze metabolites (metabolomics) predicted by the Modeling Project in wild type and knockout/overexpression strains under conditions in which phenotypes are observed, with the in vitro assigned activity facilitating both chromatographic fractionation of total metabolites and subsequent mass spectroscopic analysis; and 6) transfer in vitro enzymatic activities and in vivo metabolic functions to UniProt to update/correct the annotations for the individual binding proteins and enzymes in the metabolic pathways that are predicted and verified.

项目总结：代谢项目 代谢项目将提供实验基础设施（酶学，微生物学， 转录组学和代谢组学），用于测试用于体外预测的基于序列/结构的工具 酶活性和体内代谢功能。在使用这些工具时，新陈代谢项目将采取 领导开发发现新的代谢途径的综合战略，并为 加强工具。代谢项目的具体目标是：1）协调目标的选择， 通过Protein Core进行蛋白质生产，通过Ligand Discovery进行蛋白质纯化和配体筛选 项目，并通过建模项目进行整合途径作图; 2）测量 蛋白质核心产生的靶点，以验证通过建模 3）构建编码酶的基因的无效（敲除）菌株，所述酶的体外活性是 通过建模项目预测; 4）通过qRT-PCR进行靶向转录物分析，适当时， 通过RNA-Seq进行全基因组转录组学，以确认通过建模预测的预测代谢功能 5）分析野生型中建模项目预测的代谢物（代谢组学）， 在观察到表型的条件下敲除/过表达菌株，体外分配 促进总代谢物的色谱分离和随后的质谱分析的活性 6）将体外酶活性和体内代谢功能转移至UniProt， 更新/校正代谢途径中的单个结合蛋白和酶的注释， 预测和验证。