Researcher(s)
- Aaditya Joshi, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Wilfred Chen, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Input responsive dynamic control of gene expression is an important tool to build synthetic intracellular circuits. By developing a split-aptamer regulated trans-splicing system, we established a versatile platform, termed SPLICER, that significantly enhances the specificity and dynamic range of conditional gene expression. We have used multiple split aptamer pairs to drive the reconstitution of the split RNA intron demonstrating the modularity of our SPLICER system.
To define structural constraints for outputs, we extensively explored multiple splicing sites on a sfGFP reporter, using this as a proof-of-concept to expand the SPLICER platform to test its ability to control diverse functional outputs. Specifically, we applied this conditional splicing mechanism to regulate scaffold RNA (scRNA) designed to induce CRISPR-based transcriptional control. Additionally, we used SPLICER to regulate expression of two catalytic proteins: a viral protease to control protein cleavage and an antibiotic resistance marker to allow for input-based selection. We conducted optimization of SPLICER performance by controlling the expression levels of the SPLICER fragments or modulating the innate affinity of the two SPLICER fragments.
We expect that our experimental results in E. coli demonstrate that the SPLICER architecture provides robust, tunable control over a wide array of genetic outputs. Expanding beyond traditional fluorescent markers, these findings lay the groundwork for deploying smarter, safer molecular devices for targeted gene therapy and cellular control.