In Silico Design and Computational Validation of a Tri-Modular Genetic Circuit for Autonomous Detection and Bioremediation of Hexavalent Chromium in Closed-System Microcosms

Hexavalent chromium [Cr(VI)], a Group 1 IARC carcinogen, reaches up to 3.54 mg/L in Bangladesh’s BurigangaRiver (35-fold above the US EPA limit) due to unregulated tannery effluent. Existing remediation is passive, lackingreal-time sensing, dose-responsive treatment, and self-termination. Furthermore, deploying engineered microorganismsin open rivers poses unquantified ecological risks.Here, we present a fully computational design and validation of a tri-modular synthetic genetic circuit in a doubleauxotrophic Escherichia coli chassis for closed-system microcosms. The orthogonal modules include: (1) a ChrB-sfGFPbiosensor (pUC19) for nanomolar Cr(VI) detection; (2) a NemA chromate reductase (pET-28a), driven by the identicalPchrB promoter, for dose-responsive Cr(VI)→Cr(III) reduction; (3) a dual-trigger holin-endolysin “Deadman” killswitch (pACYC184) enforcing programmed cell death via AND-gate logic, combining chromate depletion sensingwith a 24-hour CI434-SsrA timer repressor.Validation employs five methodologies: (i) 96-hour ODE modeling of module dynamics; (ii) comparative MichaelisMenten modeling of a hypothetical NemA∗2+ catalytic variant, using kinetic parameters extrapolated from OYE-familyprecedent [15]; (iii) structural evaluation (PDB 8BPQ) via a contact-disruption score and geometric substrateplacement, providing a consistency check for the NemA∗2+ hypothesis; (iv) a ribosome-allocation metabolic modeldemonstrating sustainability of the circuit’s ∼4.5% proteome burden; and (v) Luria-Delbrück fluctuation analysis of thelayered biocontainment. Under assumed parameters, this yields a 30-day viable escapee probability (Pescape ≈ 6×10−17)in a 1,000-liter system, safely below the 10−15 threshold.Sensitivity analysis reveals this safety margin is robust to a 10× error in mutation rate or a 10× reactor scale-up, butfails at a 100× mutation rate error (Pescape = 6 × 10−15). Global Latin Hypercube Sampling (N = 10,000) across8 parameters yields median Pescape = 1.7 × 10−17, but 20.6% of samples exceed the 10−15 threshold underplausible joint-tail parameter combinations — indicating the containment margin is not yet robust and is contingenton empirical measurement of HGT transfer rates.This system computationally achieves >99% reduction of 100 µM Cr(VI) within 48 hours. Ultimately, this frameworkestablishes a transparent in silico biosafety validation template for heavy-metal bioremediation, mathematicallyquantifying both performance and mutation-driven failure modes. All work is computational; no wet-lab validationwas performed.