Multilevel Regulation in RNA-Protein Hybrid Incoherent Feed-Forward Loop Circuits for Tunable Pulse Dynamics in <i>Escherichia coli</i>

Regulating gene expression with precision is essential for cellular engineering and biosensing applications, where rapid, programmable, and sensitive control is desired. Current approaches to regulatory circuit design often rely on control at a single regulatory level, primarily the transcriptional level, thereby limiting the capability of fine-tuning the regulatory dynamics in response to complex stimuli. To address this challenge, we developed four novel RNA-protein hybrid type-1 incoherent feed-forward loop (I1-FFL) circuits in <i>Escherichia coli</i> that integrate transcriptional and translational regulators to achieve multilevel control of gene expression. These hybrid circuits leverage the modularity and rapid dynamics of RNA-based activators alongside the versatile inhibition capabilities of the protein-based repressors to endow tunable pulse dynamics through engineered delays that act as transient repressor decoys. By repurposing synthetic RNA regulators at multiple regulatory levels together with aptamers and RNA-binding proteins, we demonstrate previously unexplored circuits with tunable dynamics. Complementary simulation results highlighted the importance of the engineered delays in achieving tunable pulse dynamics in these circuits. Integrating modeling insights with experimental validation, we demonstrated the flexibility of designing the RNA-protein hybrid I1-FFL circuits, as well as the tunability of their dynamics, highlighting their suitability for applications in environmental monitoring, metabolic engineering, and other engineered biological systems where precise temporal control and adaptable gene regulation are desired.