Streamlined assembly of cloning and genome editing vectors for genus Clostridium

Tom S Bailey; Philip Hittmeyer; Yanchao Zhang; Aleksandra M Kubiak

doi:10.1016/j.isci.2023.107484

Streamlined assembly of cloning and genome editing vectors for genus Clostridium

iScience. 2023 Jul 28;26(8):107484. doi: 10.1016/j.isci.2023.107484. eCollection 2023 Aug 18.

Authors

Tom S Bailey¹, Philip Hittmeyer¹, Yanchao Zhang¹, Aleksandra M Kubiak²

Affiliations

¹ GROW - School for Oncology and Reproduction, Faculty of Health and Medical Life Sciences, Maastricht University, Maastricht, Limburg, the Netherlands.
² Exomnis Biotech BV, Oxfordlaan, Maastricht, Limburg, the Netherlands.

Abstract

Reported herein is a new set of vectors designed to streamline molecular cloning and genome editing by exploiting modern cloning methods. The new vectors build on the existing pMTL8000 vectors that have been a staple of Clostridium research for more than a decade. The introduction of two pairs of type IIS restriction sites flanking an insulated multiple cloning site in both a cloning vector and a CRISPR-Cas9 gene editing vector enables plasmid construction in a "one-pot" reaction, avoiding the more laborious steps of conventional cloning. A synthetic lacZα expression cassette introduced between the cloning sites enables visual detection of background colonies. In addition, distinct selection markers on each vector permit selection of the desired clones according to antibiotic resistance. An example of strain development using the new vectors is demonstrated.

Keywords: Biotechnology; Microbial biotechnology; Molecular Genetics.