Core--Centralized DNA preparation, fermentation and extraction facility

核心——集中DNA制备、发酵和提取设施

• 批准号: 6222379
• 负责人: peter McCarthy
• 金额: $ 10.78万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-23 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6222379
• 关键词: DNA; Streptomyces; antineoplastics; biomedical facility; drug discovery /isolation; fermentation; microorganism genetics; microorganism mass culture; microorganism metabolism; physical separation; transfection

There continues to be a need for anti-cancer agents. The discovery of novel natural products has led to the identification of several candidates which may prove to be effective anti-cancer drugs, these include the marine natural product discodermolide which was isolated from a deep water lithistid sponge. Marine microorganisms are also providing to be a source of novel natural products however little is known about secondary metabolite expression in many of these organisms. It is the goal of this program project to express the genes from these microbes in Streptomyces venezuelae. This will allow the expression of secondary metabolite gens in a well defined system, it will also allow combinatorial biology to produce unnatural natural products. This novel source of potentially bioactive metabolites will be coupled with the Novartis anti-cancer drug discovery program in order to identify new anti-cancer agents. This project includes many functions which can most efficiently be handled by a single Core facility. To this end the core laboratory at HBOI will perform the DNA extractions, fermentations and extraction of fermentation broths which are central to this project. Microorganisms will be provided to the Core laboratory from the research projects at HBOI, the University of Oregon, and UCSC for DNA extraction. The purified DNA will be shipped to the University of Minnesota where the molecular biology will be performed. The resultant clones will be shipped back to the Core laboratory where they will be fermented at a small scale, extracted and the extracts shipped to Novartis for screening. The target for throughput is 20,000 clines in year one and 30,000 in subsequent years. Clones which show activity in the Novartis screens will be refermented to confirm the production of a bioactive compound of interest. All fermentations to produce material for bioassay-guided purification of the active component will be performed by the individual research groups.

仍然需要抗癌剂。新型天然产物的发现导致了几种候选药物的鉴定，这些候选药物可能被证明是有效的抗癌药物，其中包括从深水石质海绵中分离出来的海洋天然产物discodermolide。海洋微生物也成为新型天然产物的来源，但人们对许多这些生物体中次生代谢产物的表达知之甚少。该项目的目标是在委内瑞拉链霉菌中表达这些微生物的基因。这将允许次级代谢物基因在明确的系统中表达，也将允许组合生物学产生非天然的天然产物。这种潜在生物活性代谢物的新来源将与诺华抗癌药物发现计划结合起来，以鉴定新的抗癌药物。该项目包括许多可以通过单个核心设施最有效地处理的功能。为此，HBOI 的核心实验室将进行 DNA 提取、发酵和发酵液提取，这是该项目的核心。 HBOI、俄勒冈大学和加州大学圣迭戈分校的研究项目中的微生物将提供给核心实验室用于 DNA 提取。纯化的 DNA 将被运送到明尼苏达大学进行分子生物学研究。所得克隆将被运回核心实验室，在那里进行小规模发酵、提取，并将提取物运往诺华进行筛选。吞吐量目标是第一年 20,000 株，随后几年 30,000 株。在诺华筛选中表现出活性的克隆将被转介以确认感兴趣的生物活性化合物的产生。用于生产用于生物测定指导的活性成分纯化的材料的所有发酵将由各个研究小组进行。