Temperature dependent subcellular localization

1264051 Park，谢尔顿J.温度敏感（ts）突变体广泛用于功能研究，但产生起来很麻烦，因为每种靶蛋白需要单独的文库筛选。使用高通量酵母检测，我们将工程化ts内含肽突变体，仅在允许的温度下才有活性。通过表征工程内含肽突变体，我们将开发基于温度敏感性的理解的机制。工程化的ts内含肽将用于设计结构上独立的结构域，其可以与异源蛋白质遗传融合，以温度依赖性方式将其发送到不同的亚细胞区室。用温度控制蛋白质的定位可用于调节它们的体内活性并产生有用的条件突变体。我们将通过以温度依赖性方式从细胞质到细胞核靶向模型转录因子rtTA和Gal 4来展示设计，并表明它们的转录活性与它们的核靶向相关。我们还将表明，细胞质激酶，Ste 7，可以本地化到不同的细胞隔室使用工程内含肽。所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质的定位和功能，并简化基于简单克隆而不是文库筛选的新ts突变体的工程化。Ts突变体通常用于研究，但难以预测地进行工程改造。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ts内含肽突变体来设计一个模块化的靶向结构域，该结构域可以与异源蛋白融合，以温度依赖性方式调节其亚细胞定位。温度敏感条件突变体的简易构建将有助于在体内研究蛋白质功能。PI在部门，学校和大学层面参与了广泛的举措，以改善大学预科，本科和研究生教育。目前的赠款将支持继续参与这些推广计划，并为K-12和本科生提供额外的研究机会。将有新的教材开发的基础上提出的实验，以推进工程教育在实验室内外。通过实施所描述的实验，学生将学习基本的合成生物学的基础上模块化建设的生物成分。这个奖项由生物技术，生物化学，生物质工程计划的CBET部门是共同资助的系统和合成生物学计划的分子和细胞生物学部门。

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.