Automated design and implementation of a NOR gate in Pseudomonas putida

Boolean NOR gates have been widely implemented in Escherichia coli as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and the metabolic engineering platform Pseudomonas putida with components tailored for E. coli. To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library three inducible promoters that are specific to P. putida, thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from E. coli to P. putida, the CELLO software was still able to find an effective NOR gate in the new chassis. The automated generation of the corresponding DNA sequence and in vivo experimental verification accredited that some genetic modules initially optimized for E. coli can indeed be reused to deliver NOR logic in P. putida as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for the quick assembly of genetic circuits to meet complex specifications.