Streamlined assembly of cloning and genome editing vectors for genus Clostridium

Tom S. Bailey*, Philip Hittmeyer, Yanchao Zhang, Aleksandra M. Kubiak

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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 lacZa 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.
Original languageEnglish
Article number107484
Number of pages14
JournaliScience
Volume26
Issue number8
DOIs
Publication statusPublished - 18 Aug 2023

Keywords

  • Biotechnology
  • Microbial biotechnology
  • Molecular Genetics

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