A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations.

ABSTRACT: Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227-bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10% of colonies, results in a truncation of ld

SUBMITTER: Dierksheide KJ 

PROVIDER: S-EPMC10802373 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature