Protocol for applying SUPER to suppress leakage in layered toehold-switch circuits and stabilize kill switches in Escherichia coli

RNA-based regulators are versatile building blocks for synthetic circuits, but the narrow dynamic range and leaky expression often limit performance. Here, we present SUPER, a framework that upcycles an existing regulator by designing a programmable synthetic small RNA (sRNA) as a post-transcriptional add-on controller, without modifying the existing device. Using a toehold switch as a worked example in E. coli , we describe cognate sRNA construction, multi-plasmid transformation, flow cytometry characterization, riboregulator tuning, and stabilization of a genetic kill switch. For complete details on the use and execution of this protocol, please refer to Heo et al. 1