Dynamics of genetic circuits in Pseudomonas protegens

Abstract

The engineering of genetic circuits to perform predefined computations is central to synthetic biology, enabling living cells with new functionalities applicable across various domains. However, these circuits are often specifically tailored to particular cellular hosts, with Escherichia coli being the most popular. Consequently, their intended functions may not translate well to other organisms, limiting their scope. Understanding circuit dynamics in less familiar organisms is crucial, especially for niche-specific applications requiring cellular chassis different from model organisms generally used in synthetic biology. Here, we develop a combined experimental and theoretical pipeline to evaluate the performance of NOT logic circuits, also called inverters, in the soil bacterium Pseudomonas protegens Pf-5--a host renowned for its unique environmental functions and a newcomer to genetic circuitry. Inverters were experimentally tested to characterize input-output functionality, and mathematical modelling was used to infer the dynamic principles of circuit modules. The model quantified the individual impacts of key parameters--such as translation efficiency, repressor performance, and promoter activity--on output levels, enabling predictions about inter-host circuit portability. This parameter calibration revealed unique properties of the chassis, including faster transitions between on and off circuit states compared to the synthetic biology workhorse Pseudomonas putida. These characteristics may reflect adaptations to the fluctuating conditions of the plant rhizosphere, where this bacteria thrives. As a result, our work provides DNA parts, circuits and mathematical characterizations to establish P. protegens Pf-5 as a viable chassis for environmental synthetic biology.