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.

项目摘要：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EFI的基于序列/结构的工具，用于预测体外 酶促活性和体内代谢功能。在使用这些工具时，代谢项目将采用 领导着开发综合策略，以发现新的代谢途径并为 增强工具。代谢项目的具体目的是：1）协调目标的选择 通过蛋白质核，蛋白质纯化和配体筛选的蛋白质生产。 项目和集成途径映射由建模项目； 2）测量体外酶促活性 蛋白质核产生的靶标通过建模验证整合途径映射的预测 项目; 3）构建了编码体外活性的酶的基因构造（基因敲除）菌株 由建模项目预测； 4）通过QRT-PCR执行有针对性的成绩单分析，并在适当的情况下进行 RNA-Seq全基因组转录组学确认通过建模预测的预测代谢功能 项目; 5）分析野生类型的建模项目预测的代谢产物（代谢组学） 在观察表型的条件下，敲除/过表达菌株，并分配了体外 促进总代谢物的色谱分馏和随后的质谱 分析; 6）体外酶活性和体内代谢功能转移至单体 在代谢途径中更新/纠正单个结合蛋白和酶的注释 被预测和验证。