Streamlined assembly of cloning and genome editing vectors for genus Clostridium

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. [Display omitted] •One-pot cloning with 97%–99% accuracy•One-pot transfer of subcloned expression cassettes to a genome editing vector•Transciptionally insulated cloning sites precludes the need to clone terminators•The new vectors enable standardized cloning in tubes or microplates Molecular Genetics; Biotechnology; Microbial biotechnology