Temperature dependent subcellular localization

1264051Park，Sheldon J.温度敏感(Ts)突变体被广泛应用于功能研究，但由于每个目标蛋白都需要单独的文库筛选，因此产生起来很麻烦。使用高通量酵母菌检测，我们将对仅在允许温度下有效的突变株进行工程改造。通过表征工程内含子突变体，我们将发展一种基于对温度敏感性的理解的机制。基因工程的ts整合素将被用来设计一个结构独立的结构域，该结构域可以与异源蛋白在基因上融合，以温度依赖的方式将其发送到不同的亚细胞隔间。用温度控制蛋白质的定位可能有助于调节它们在体内的活性并产生有用的条件突变。我们将以温度依赖的方式将模型转录因子RTTA和Gal4从细胞质靶向细胞核，并证明它们的转录活性与它们的核靶向性相关。我们还将展示细胞质激酶Ste7可以使用工程内含子定位于不同的细胞隔间。设计的靶向模块将允许使用生长温度动态控制细胞内蛋白的定位，从而实现功能的动态控制，并简化基于简单克隆而不是文库筛选的新ts突变体的工程。TS突变体通常用于研究，但很难进行可预测的工程。温度敏感性的物理基础也没有得到很好的理解。我们将设计一种模块化的靶向结构域，可以与异源蛋白融合，以温度依赖的方式调节其亚细胞定位。温度敏感型条件突变体的简便构建将为体内蛋白质功能的研究奠定基础。PI参与了系、校和大学层面的广泛倡议，以改善大学预科、本科生和研究生教育。目前的拨款将支持继续参与这些外展项目，并为K-12和本科生提供更多的研究机会。将根据拟议的实验开发新的教材，以促进实验室内外的工程教育。通过实施上述实验，学生将学习基于生物成分模块化结构的基本合成生物学。该奖项由CBET部门的生物技术、生化和生物质工程项目获得，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

1264051Park, Sheldon J. Temperature sensitive (ts) mutants are widely used in functional studies but are cumbersome to generate, since a separate library screening is needed for each target protein. Using high throughput yeast assays, we will engineer ts intein mutants that are only active at a permissive temperature. By characterizing engineered intein mutants, we will develop a mechanism based understanding of temperature sensitivity. The engineered ts intein will be used to design a structurally independent domain that can be genetically fused to a heterologous protein to send it to different subcellular compartments in a temperature dependent manner. Controlling the localization of a protein with temperature may be useful to regulate their in vivo activity and generate useful conditional mutants. We will demonstrate the design by targeting model transcription factors, rtTA and Gal4, from the cytoplasm to the nucleus in a temperature dependent manner, and show that their transcriptional activities correlate with their nuclear targeting. We will also show that the cytoplasmic kinase, Ste7, can be localized to different cellular compartments using engineered inteins. The designed targeting module will allow dynamic control of the localization, and hence the function, of intracellular proteins using the growth temperature, and simplify the engineering of new ts mutants based on simple cloning rather than library screening. Ts mutants are commonly used in research but are difficult to engineer predictably. The physical basis of temperature sensitivity is also not well understood. We will engineer ts intein mutants to design a modular targeting domain that can be fused to a heterologous protein to regulate its subcellular localization in a temperature dependent manner. Facile construction of temperature sensitive conditional mutants will be useful for studying protein function in vivo. The PI participates in a broad range of initiatives at the department, school, and university levels to improve pre-collegiate, undergraduate, and graduate education. The current grant will support continued participation in these outreach programs, and provide additional research opportunities to both K-12 and undergraduate students. There will be new teaching materials developed based on the proposed experiments to advance engineering education both in and out of the lab. By implementing the described experiments, the students will learn essential synthetic biology based on modular construction of biological components.This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.