Dynamics of genetic circuits in Pseudomonas protegens

Genetic circuit engineering enables new cellular functions, yet most circuits are developed in the model host Escherichia coli, limiting their availability and performance in alternative organisms. To expand chassis options, we developed an experimental-theoretical pipeline to evaluate NOT logic circuits, or inverters, in the soil bacterium Pseudomonas protegens Pf-5, a species with valuable environmental traits and a newcomer to bioengineering. We characterized the inverter's input-output behavior and used mathematical modeling to infer key dynamic principles. The model quantified how parameters such as translation efficiency, repressor performance, and promoter activity shape circuit output and influence inter-host portability. Pf-5 displayed distinct properties, including steeper on/off transitions than the synthetic biology workhorse Pseudomonas putida. A model-guided design of two compatible inverters connected in series was validated, producing a YES logic response. This work provides DNA parts, circuits, and models that establish P. protegens Pf-5 as a promising chassis for environmental synthetic biology. A record of this paper's transparent peer review process is included in the supplemental information.