A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria

Genetically encoded logic gates,
especially invertersNOT
gatesare the building blocks for designing circuits, engineering
biosensors, or decision-making devices in synthetic biology. However,
the repertoire of inverters readily available for different species
is rather limited. In this work, a large whole of NOT gates that was
shown to function previously in a specific strain of Escherichia
coli, was recreated as broad host range (BHR) collection
of constructs assembled in low, medium, and high copy number plasmid
backbones of the SEVA (Standard European Vector Architecture) collection.
The input/output function of each of the gates was characterized and
parametrized in the environmental bacterium and metabolic engineering
chassis Pseudomonas putida. Comparisons of the resulting
fluorescence cytometry data with those published for the same gates
in Escherichia coli provided useful hints on the
portability of the corresponding gates. The hereby described inverter
package (20 different versions of 12 distinct gates) borne by BHR
plasmids thus becomes a toolbox of choice for designing genetic circuitries
in a variety of Gram-negative species other than E. coli.