FUS-Controlled Unidirectional Iron Sequestration in E. coli Nissle 1917 for Tumor Metabolic Collapse SECTION 1: ABSTRACT Solid tumors establish a metabolically distinct microenvironment (TME) characterized by hypoxia and necrosis—conditions that selectively favor the colonization of E. coli Nissle 1917 (EcN) (Stritzker et al., 2007). Once established, the bacteria encounter a niche defined by elevated lactate levels (Pérez-Tomás & Pérez-Guillén, 2020) and an intense demand for iron by cancer cells. While EcN typically upregulates enterobactin biosynthesis to compete for these resources, the host protein Lipocalin-2 neutralizes this siderophore, effectively limiting bacterial iron scavenging (Huang et al., 2024). This project engineers EcN to produce salmochelin—a Lipocalin-2-resistant siderophore (Fischbach et al., 2006) encoded by the iroBCDE/iroN locus, which is a key determinant of EcN’s competitive survival in iron-restricted environments (Massip et al., 2019). To implement this therapeutic logic, a FUS-controlled genetic circuit was designed in Asimov Kernel and digitally assembled in Benchling using the native pMUT2 backbone of EcN (CP023342.1). The circuit integrates a thermal activation cassette (TlpA39C/pTlpA) triggered by Focused Ultrasound, a lactate-gated lysis kill switch (BBa_K3848004) for tumor-specific biocontainment, and dual sRNA effectors targeting fur and iroN to drive unidirectional iron sequestration, that depletes the tumor’s labile iron pool, inducing cancer cell metabolic collapse (Pinnix et al., 2010; Saha et al., 2019). Boolean circuit logic was computationally simulated in Python and the complete 10,114 bp therapeutic vector was assembled in silico via Gibson Assembly at the s2 intergenic site of pMUT2, ensuring antibiotic-free stability through the endogenous RelB/RelE toxin-antitoxin system (Kan et al., 2021).