Streamlining marker-less allelic replacement in Streptococcus pneumoniae through a single transformation step strategy: easyJanus.

ABSTRACT: The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic exchange predominantly using the kan^R-rpsL cassette known as "Janus." The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene's flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target's downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragme

SUBMITTER: Chembilikandy V 

PROVIDER: S-EPMC11409681 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature