Coupling environmental biosensors to inverter circuits in soil bacteria

Genetic logic gates, such as inverting NOT functions, are routinely designed and optimised for a narrow set of laboratory-standard chemical inputs. This reliance limits their utility in real-world applications, where engineered chassis must navigate complex, niche-specific signals. Focusing on environmental synthetic biology, we engineered four biosensors responsive to ecologically relevant physical and chemical inputs (iron, oxygen, temperature, and light) and systematically coupled them to three repressor-based NOT logic gates (QacR, SrpR, and LitR) to generate 12 composite sensor-gate circuits. To evaluate cross-host robustness, all constructs were benchmarked in two non-model soil bacteria: Pseudomonas putida KT2440 and Pseudomonas protegens Pf-5. Our findings demonstrate that overall circuit performance is non-additive and cannot be predicted simply by summing individual sensor and gate dynamics. While standalone biosensors reliably detected their cognate inputs, concatenation across regulatory junctions introduced non-linearities, emergent dynamics, and host-dependent effects that caused several pairings to deviate unexpectedly from their predicted behaviour. Furthermore, host context directly governed logic execution: P. protegens Pf-5 exhibited superior overall circuit-processing capacity, whereas specific configurations, such as iron-responsive gates, performed better in P. putida KT2440. All 12 constructs are modularly integrated using the Standard European Vector Architecture (SEVA) format, providing a benchmarked toolkit to program complex information processing in real-world environments.