Synthetic tools for deep engineering of Pseudomonas putida as a surface display chassis

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Esteban Martinez Garcia, Sofia Fraile and Victor de Lorenzo

Centro Nacional de Biotecnologia (CNB-CSIC), Spain

While Systems Biology aims at acquiring a global knowledge of the physiology of the cell, Synthetic Biology pursues inter alia the reprogramming of organisms to execute new-to-nature functions. Deep engineering tasks need to optimize two factors to maximize reprogramming efficiency. First, it is fundamental to possess a suitable genetic tool repertoire and second is to select an appropriate chassis that is easy to manipulate genetically and offers good biotechnological properties. On this background we have developed a number of molecular assets consisting of a collection of constructs assembled in a modular fashion to effortlessly exchange the different functional parts at users’ convenience denominated Standard European Vector Architecture (SEVA). To this end, we have produced dedicated vectors to both eliminate undesired chromosomal regions or –on the contrary, stably implant genetic networks. In order to facilitate the use of these tools, users can interrogate a vector repository to find an optimal configuration through the website A large number of intrinsic traits make the soil bacterium Pseudomonas putida an optimal choice as a chassis of reference for different environmental and biotechnological purposes. It is a non-pathogenic, ubiquitous bacterium with a broad metabolic versatility with a considerable tolerance to organic solvents and xenobiotic chemicals. This makes P. putida an appealing organism to develop platform strains for Synthetic Biology. To this end, we have used different SEVA tools for editing the genome of P. putida KT2440 in order to create a distinct type of chassis (x-S) that is enhanced for surface display of proteins. This consists of a cell deleted of its social determinants and designed for presenting active proteins to the external medium that are still attached to the bacterial body. The applications of such strain for creating artificial communities and for acting as a live scaffold for an exo-reactor will be presented.