SGER: Quantitating Low-Copy-Number Proteins in Individual Cells Using Microfluidics and Single-Molecule Counting

点击翻译按钮获取中文摘要

The last ten years have witnessed great advances in analytical instrumentation that have revolutionized our way of studying living systems and significantly improved our understanding of these systems. The genome sequencing and DNA microarray technology are breakthroughs that demonstrate the remarkable power of new instrumentation. They have allowed massive amounts of data to be generated on interesting and relevant systems. Much of this data is collected from thousands of cells simultaneously. Microfluidics techniques can be used to manipulate single cells as well as applied to other analysis tasks involving minute quantities of samples. It is also known that single molecule techniques can allow precise quantification and analysis of individual molecules. Recently, the principle investigator has developed a device that combines single molecule detection with microfluidic manipulation to obtain absolute quantification of phycobiliprotein subcomplexes within a single cyanobacterial cell, which can exist at low copy numbers. This SGER project first focuses on improving the performance and generality of the single cell analysis device. Then this device will be used to characterize how the cell-to-cell variation affects evolutionary selection and biological diversity of cyanobacteria. Information obtained is crucial to the future understanding of complex biological systems. Instead of examining cell homogenates, one can investigate single cells and understand how each cell differs from one another. Consequently, the development of such an instrument will be of great general interest to those who study the biology of cells. The intellectual merit of this approach is that it provides the ultimate analytical tool for quantifying rare-copy-number proteins cell by cell, and that it can reveal how the group behavior of cells are related to cell-to-cell variations.Broader Impacts:The device developed in this project will be a general platform for quantitative analysis (down to the single molecule level) of biomolecules in a single cell. It will significantly improve the accessibility of microfluidic and single molecule methodology to the scientific community. This research includes state-of-the-art training of graduate students with microfluidic fabrication, chemical analysis, electronics, programming, and microscopy.