Synthetic receptors are powerful tools for cellular engineering, yet their utility is often constrained by constitutive activity and the absence of natural feedback mechanisms that maintain cellular homeostasis. To address this, we engineered an autonomous regulatory circuit for synthetic Notch (synNotch) receptor activation using an orthogonal microRNA (miRNA)-mediated negative feedback mechanism in mammalian cells. Specifically, synNotch activation induces the expression of a synthetic miRNA that targets complementary sites engineered into the synNotch transcript, resulting in a self-limiting feedback loop. We demonstrate that synNotch repression positively correlates with the number of miRNA target sites integrated into the receptor transcript, enabling tunable control of receptor expression. Furthermore, flow cytometry and live-cell imaging analyses revealed effective suppression of synNotch expression following activation. Downstream target gene expression peaked at approximately 48 hours post-activation before gradually declining as the feedback circuit engaged. This approach achieves autonomous synthetic receptor regulation-eliminating the need for external intervention-and enables tunable response dynamics through rational circuit design. Our miRNA feedback strategy provides a generalizable platform for engineering self-regulating synthetic receptor systems with improved control and predictability for cellular engineering applications.
Crisis support teams’ technological openness and learning attitudes toward the AI based virtual patient system crisis support VR
BackgroundAgainst the backdrop of escalating global humanitarian crises, innovative didactic simulations are becoming increasingly important. A promising alternative to traditional classroom-based didactics for learning psychological