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

蓝藻是少数几种形成内部结构的细菌之一，被称为脂滴，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。当细胞生长减慢时，这些液滴会在细胞内增加，但人们对它们的相关蛋白质、它们是如何形成的，以及它们的生理功能几乎一无所知。本研究将确定与这些结构相关的蛋白质，并通过改变这些蛋白质的表达和观察其产生的影响来推断这些蛋白质的功能。除了为未知功能的基因分配角色外，这项工作还将扩展我们对这些光合生物中鲜为人知的结构的理解。该项目将吸引本科生和硕士生（包括来自科学领域代表性不足群体的学生）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，学生将在首席研究员的指导下学习计划和进行实验，并解释实验数据。在教学实验室，学生将进行为期一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脂滴相关的蛋白质，并根据其与其他系统中脂滴中发现的蛋白质的相似性，确定其他蛋白质的研究目标。假定的脂滴相关蛋白将使用荧光蛋白融合标记和测试定位到脂滴。一个利用脂滴蛋白质组蛋白的细菌2杂交系统将被用作从肽库中筛选其他相互作用蛋白的诱饵。其次，每个鉴定的蛋白质的作用将通过过表达和突变实验确定。将筛选得到的菌株在不同碳/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与野生型的表型改变。特定突变体和过表达菌株将根据结构、分离的脂滴组成、相关的酶活性或光合能力进行表征，以确定这些蛋白质的功能和脂滴在蓝藻中的作用。

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.