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Full Stream Name: Light Switches for Protein Function

Can we use optogenetics to eliminate proteins of interest in vivo in order to learn their functions?

Much of what we know about Biology has relied on our ability to generate loss-of-function mutants, which provide insight into the functions of the affected genes and their protein products. One impediment to learning the diverse functions of specific proteins occurs when they are required for organism viability, making it impossible to generate viable homozygous mutant individuals. Recent advances in our understanding of light-responsive proteins and of the cellular machinery for protein degradation provide an opportunity to develop a general method for conditional, spatially- and temporally-controlled, light-dependent (optogenetic) degradation of proteins of interest. The approach being developed utilizes two plant proteins, CRY2 and CIB1, which bind one another in response to blue light illumination. We are testing the ability of several CRY2 fusion proteins to interact with and degrade CIB1 fused to two test proteins, GFP (green fluorescent protein) and the Drosophila developmental protein, Cactus. Students in this stream will isolate, map, and characterize transgenic flies that carry CRY2 fused to one of six ubiquitin ligases (determinants of protein degradation). Depending on their progress and timeline, students can further participate in testing the ability of their CRY2-ubiquitin ligases to direct light-dependent degradation of CIB1-GFP and CIB1-Cactus in fly embryos. Over the course of this work, students will gain understanding of both classical and molecular genetic analysis of fruit flies and their practical applications, as well as an understanding of Drosophila embryonic development.

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