RUI: Determination and Functional Analysis of the Cyanobacterial Lipid Droplet Proteome

RUI：蓝藻脂滴蛋白质组的测定和功能分析

• 批准号: 1413583
• 负责人: Michael Summers
• 金额: $ 62.81万
• 依托单位: The University Corporation, Northridge
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413583&HistoricalAwards=false
• 关键词: RUI; Determination; Functional; Analysis; Cyanobacterial

Cyanobacteria are amongst the few bacterial species that form the internal structures known as lipid droplets, which are of interest to the biotechnology industry. These droplets could supply material for food, fuel and the chemical and pharmaceutical industries. These droplets increase in the cell when growth slows, but almost nothing is known about their associated proteins, how they are formed, or their physiological function. This study will identify the proteins associated with these structures, and infer the function of these proteins by altering their expression and observing the resulting effects. In addition to assigning roles to genes with unknown function, this work will extend our understanding of a poorly understood structure in these photosynthetic organisms. This project will engage undergraduate and Master's level students (including students from groups under-represented in science) in research projects in molecular genetics, biochemistry, and bacterial physiology. In the research laboratory, students will learn to plan and conduct experiments and to interpret experimental data under the mentorship of the principal investigator. In the teaching laboratory, students will conduct semester-long original research projects designed to teach the theory and practice of molecular biology.The overall goal of this Research at Undergraduate Institutions project is to understand the function and formation of cyanobacterial lipid droplets. This will be accomplished as a result of two specific objectives. First, the proteins associated with purified lipid droplets will be identified by mass spectrometric analysis, and additional proteins will be targeted for study based upon their similarity to proteins found in lipid droplets in other systems. Putative lipid droplet associated proteins will be tagged using fluorescent protein fusions and tested for localization to lipid droplets. A bacterial 2-hybrid system using proteins from the lipid droplet proteome will be used as bait to screen for additional interacting proteins from a peptide library. Second, the role of each identified protein will be experimentally determined by over-expression and mutation. The resulting strains will be screened for phenotypic alterations from wild-type with regard to growth under various carbon/nitrogen/light conditions, lipid droplet morphology, and the ability to resume growth following stationary phase or stress. Specific mutants and over-expression strains will be characterized with regard to structure, lipid composition of isolated droplets, and associated enzymatic activities or photosynthetic abilities to determine the function of these proteins and role of lipid droplets in cyanobacteria.

蓝细菌是为数不多的形成称为脂滴的内部结构的细菌物种之一，这是生物技术行业感兴趣的。 这些液滴可以为食品、燃料、化学和制药工业提供材料。当细胞生长减缓时，这些液滴会在细胞中增加，但对它们的相关蛋白质、它们是如何形成的或它们的生理功能几乎一无所知。这项研究将确定与这些结构相关的蛋白质，并通过改变它们的表达和观察所产生的影响来推断这些蛋白质的功能。 除了为功能未知的基因分配角色之外，这项工作还将扩展我们对这些光合生物中人们知之甚少的结构的理解。 该项目将吸引本科和硕士水平的学生（包括来自科学代表性不足的群体的学生）参与分子遗传学，生物化学和细菌生理学的研究项目。在研究实验室，学生将学习计划和进行实验，并在首席研究员的指导下解释实验数据。 在教学实验室，学生将进行为期一个学期的原创性研究项目，旨在教授分子生物学的理论和实践。本科院校研究项目的总体目标是了解蓝藻脂滴的功能和形成。这将通过两个具体目标来实现。首先，将通过质谱分析鉴定与纯化的脂滴相关的蛋白质，并基于其与其他系统中脂滴中发现的蛋白质的相似性，将其他蛋白质作为研究的目标。将使用荧光蛋白融合物标记推定的脂滴相关蛋白，并测试脂滴的定位。 使用来自脂滴蛋白质组的蛋白质的细菌双杂交系统将用作诱饵以从肽文库中筛选另外的相互作用蛋白质。 第二，每种鉴定的蛋白质的作用将通过过表达和突变来实验确定。将针对在各种碳/氮/光条件下的生长、脂滴形态以及在稳定期或胁迫后恢复生长的能力方面相对于野生型的表型改变来筛选所得菌株。特定的突变体和过表达菌株的特征在于关于分离的液滴的结构，脂质组成，和相关的酶活性或光合能力，以确定这些蛋白质的功能和脂滴在蓝藻中的作用。