Construction of pDYN6902, a new Streptomyces integrative expression vector designed for cloning sequences interfering with Escherichia coli viability

Annabelle Thibessard (1) , Claire Bertrand (1) , Julien Hiblot (2, 1) , Emilie Piotrowski (1) , Pierre Leblond (1)

1 DynAMic - Dynamique des Génomes et Adaptation Microbienne
2 EPFL - Ecole Polytechnique Fédérale de Lausanne

We describe here the construction of a new version of the Escherichia coli–Streptomyces shuttle cloning vector pIJ6902, which carries the original thiostrepton-inducible PtipA as well as the integrative and cloning systems, but with the E. coli low copy number replication control of the F (fertility) factor. The rationale of this construct was to provide a shuttle cloning vector to enable the cloning of genes whose expression is toxic in E. coli while keeping the efficient regulated expression system for Streptomyces as its ascendant vector pIJ6902. This new vector named pDYN6902 (10,975 bp) showed a copy-number reduced 12 times compared to that of pIJ6902 and indeed a decreased expression of the cloned gene. It was also shown to allow the successful cloning in E. coli of a deleterious gene, i.e. I-SceI meganuclease encoding gene.

Mots clés: Toxic; tipA promoter; Cloning; Escherichia coli; Streptomyces

Domaines: Biotechnologies

Annabelle Thibessard, Claire Bertrand, Julien Hiblot, Emilie Piotrowski, Pierre Leblond. Construction of pDYN6902, a new Streptomyces integrative expression vector designed for cloning sequences interfering with Escherichia coli viability. Plasmid, 2015, 82, pp.43-49. ⟨10.1016/j.plasmid.2015.10.003⟩. ⟨hal-01258339⟩