SUPER: Upcycling Genetic Parts for Precise Gene Expression Control, Leakage Minimization, and Genetic Circuit Stability

Abstract A long‐standing goal of synthetic biology is to reprogram cells by rewiring genetic parts. Despite the expanding library of genetic parts, construction of integrated synthetic circuits with desired specifications remains challenging in part due to intricate dependence on sequence contexts, where unexpected narrow dynamic ranges and leaky expression can plague system performance. To provide an alternative approach to the screening process of iterative design‐build‐test cycles, SUPER ( S ynthetic U pcycling P latform for E ngineering R egulators), a modular platform for upcycling genetic devices is introduced. Inspired by antagonistic regulation mechanisms, SUPER employs small RNA as an add‐on controller to modulate gene expression patterns without genetic modification of target regulators. SUPER not only enhances the performance of RNA‐, chemical‐, temperature‐, and protein‐responsive regulators up to 1011%, but also allows to cover an expanded dynamic range up to 22 018.9‐fold. This enhanced control can provide genetic circuit stability, particularly under strong selective pressures, as demonstrated with a Holin‐expressing kill switch integrated with SUPER, maintaining stable functionality for over 30 days. Finally, SUPER combines with an environmental sensor, TlpA36, functioning as a chemical‐ and temperature‐responsive 2‐input kill switch. Featuring straightforward design, minimal cellular burden, and expanded tunability, SUPER provides a systematic upcycling framework for genetic circuit construction in biotechnology.