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

## TL;DR

A 227-bp deletion in a commonly used Bacillus subtilis vector causes unintended gene mutations that may go unnoticed in experiments.

## Contribution

Identifies a historical deletion in a vector that leads to undetected loss-of-function mutations in downstream genes.

## Key findings

- A 227-bp deletion in the amyE integration vector causes unintended recombination in ~10% of colonies.
- The deletion leads to a truncation of the ldh gene, which may affect fermentative metabolism undetected.
- Both correct and incorrect recombinations test positive for amyE disruption, potentially confounding experiments.

## 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 ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.