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.
