Broadening the SEVA Plasmid Repertoire to Facilitate Genomic Editing of Gram-Negative Bacteria

Abstract

Editing bacterial genomes to a predetermined extent is essential for many purposes of the synthetic biology and biotechnology fields. The use of the I-SceI deletion system has proven fundamental for such tasks in the Gram-negative bacterium Pseudomonas putida. Moreover, we have developed the Standard European Vector Architecture (SEVA) repository that comprehends a collection of plasmids that allows performing a wide number of different molecular operations. The limited number of antibiotic selection markers of the pSW-plasmids pointed us to SEVA-rize the pSW-I plasmid to expand its functionality and use. We have named these new variants as pSEVAn28S, being n any of the six different antibiotic markers of the SEVA plasmids: Ap, Km, Cm, Sm/Sp, Tet, and Gm. In that way, we have updated the previous I-SceI deletion system in two ways: (1) implementing isothermal assembly to avoid the use of restriction enzymes and T4 DNA ligase within this protocol to assemble the plasmids with the respective TSs and (2) standardizing and expanding the I-SceI-expressing plasmid variants. In this protocol, we explain the use of isothermal assembly to construct the non-replicative plasmid bearing the chromosomal homology regions and the use of different pSEVAn28S plasmids to induce the expression of the I-SceI endonuclease to obtain a deleted strain in Gram-negative bacteria.