Genetic controllers for enhancing the evolutionary longevity of synthetic gene circuits in bacteria

Engineered gene circuits often degrade due to mutation and selection, limiting their long-term utility. Here we present designs for genetic controllers which maintain synthetic gene expression over time. Using a multi-scale “host-aware" computational framework, which captures interactions between host and circuit expression, mutation, and mutant competition, we evaluate several controller architectures based on three metrics for evolutionary stability: total protein output, duration of stable output, and half-life of production. We propose a number of designs with varying inputs (e.g., output per cell, growth rate) and actuation methods (transcriptional vs. post-transcriptional regulation). We find post-transcriptional controllers generally outperform transcriptional ones, but no single design optimizes all goals. Negative autoregulation prolongs short term performance, while growth-based feedback extends functional half-life. We propose three biologically feasible, multi-input controllers that improve circuit half-life over threefold without requiring coupling the process to an essential gene or a genetic kill switch.