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

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.