RUI: Complex control in C. elegans using de novo designed protein switches

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A fundamental goal in synthetic biology is to control cellular process via the targeted degradation of important regulatory proteins. The aim of this project is to develop and test a specific and tunable system for protein degradation in a model eukaryotic organism. This project is viewed as an important step towards the application of this tool in more complex biological systems and ultimately towards its application in biomedical research and biotechnology. This project will engage a diverse group of undergraduate student researchers in interdisciplinary research during the academic year and summer, improving their creative problem-solving skills, independent thinking, and ability to communicate with both scientific audiences and the general public. Portions of this research will be integrated into the Molecular Genetics and Biochemistry courses at Wabash College, engaging a large number of students in novel scientific discovery. Current methods of controlling essential and pleiotropic proteins in C. elegans suffer from limitations, including incomplete penetrance, false negatives, and the inability to differentially control multiple genes with the same system simultaneously. The project team will pioneer the Latching Orthogonal Cage/Key pRoteins (LOCKR) method of protein control in C. elegans. LOCKR encompasses a de novo designed protein “Switch” capable of caging a linear bioactive motif and an inducible peptide “Key.” The peptide Key unlocks the caged motif in the Switch, eliciting its bioactive function. The LOCKR system’s de novo origins allow the design of multiple Switches and Keys as orthogonal pairs to control multiple proteins simultaneously. A nematode-optimized degronLOCKR Switch (NemDegronLOCKR) will afford C. elegans researchers a robust protein control method with tissue specificity and the ability to independently control multiple genes at various time points in development. This project applies computational design, in vitro biophysical analysis, and in vivo experiments using CRISPR-Cas9 to fuse NemDegronLOCKR to genes of interest. The results of this study will be broadly disseminated by the project team through presentations and publications. C. elegans strains and DNA constructs will be deposited at the Caenorhabditis Genetics Center and Addgene, respectively. The LOCKR system is highly tunable, as virtually any linear motif can be caged. The flexibility of the LOCKR system, and its ability to control signaling pathways and cellular targeting, will support synthetic biologists seeking to produce novel lifelike systems. Demonstration of the utility of the LOCKR system in nematodes lays a firm foundation for its use in other, even more complex organisms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.