UNS: Engineering protein sensors to detect epigenetic changes during stem cell culture

DNA甲基化在调节基因活性中起着重要作用，在细胞群体中，细胞可能表现出不同水平的甲基化，从而导致不同于群体平均水平的行为。了解单个细胞中甲基化水平的变化在许多生物学应用中是重要的。在这个项目中，研究人员将开发一种新的方法，可以在单细胞水平上测量甲基化。智力优势：目前用于量化DNA甲基化的检测方法是有限的，因为它们排除了单细胞分析和表观遗传变化的实时监测。研究人员建议将蛋白质工程原理与现代荧光检测技术相结合，设计一种探针。将追求三个目标：设计和验证用于检测基因位点附近甲基化DNA的分子传感器，优化用于细胞提取物的探针，并在活细胞中评估遗传编码探针。此外，还将测量干细胞传代过程中甲基化水平的变化。更广泛的影响：该探针能够显著提高单细胞DNA甲基化水平的检测能力，并有可能监测动态变化。该研究项目为工程师提供了一个多学科的培训机会。这些项目将让本科生参与研究，并鼓励少数民族和女学生的参与。该提案还将支持与工程项目中的女性继续合作，并开发一个新的实验室模块，以吸引学生了解基于荧光的检测技术的概念。

1512285 Yuan, Chongli DNA methylation is important in regulating gene activity , and in a cell population cells may have exhibit different levels of methylation, which results in behaviors that vary from the population average. Understanding the methylation level changes in individual cells is important in many biological applications. In this project the researchers will develop a novel method which will enable measurement of methylation at a single-cell level. Intellectual Merit: Current detection approaches for quantifying DNA methylation are limited because they preclude single-cell analysis and real-time monitoring of epigenetic changes. The researchers propose to design a probe by combining protein engineering principles and modern fluorescence detection techniques. Three objectives will be pursued: engineer and validate molecular sensors that detect methylated DNA near a gene locus, optimize the probes for use with cell extracts, and evaluate the genetically-encoded probe within live cells. Additionally, methylation level changes during stem cell passaging will also be measured. Broader Impacts: The proposed probe has the capability to significantly improve detecting DNA methylation levels at a single cell and potentially enable monitoring of dynamic changes. The research project provides a multidisciplinary training opportunity for engineers. The PIs will involve undergraduate students in research and encourage the participation of minority and female students. This proposal will also support the continued collaboration with the Women in Engineering Program and develop a new laboratory module for engaging students with the concept of fluorescence-based detection techniques.