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Volume 170, Issue 4

A historical sequence deletion in a commonly used chromosome integration vector generates undetected loss-of-function mutations Open Access

Abstract

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering . One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the locus. Here, we report a gap in the homology region inherited from the original 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 and the downstream gene , 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 , which encodes lactate dehydrogenase. Although both types of colonies test positive for disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): amyE , Bacillus subtilis , fermentation , integration vector and pDG1661
Funding
This study was supported by the:
  • National Science Foundation (Award CAREER Award MCB-1844668)
    • Principle Award Recipient: Gene-WeiLi
  • National Institutes of Health (Award R35GM124732)
    • Principle Award Recipient: Gene-WeiLi
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2024-04-11
2024-04-29
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A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations
Microbiology 170, 001455 (2024); https://doi.org/10.1099/mic.0.001455
/content/journal/micro/10.1099/mic.0.001455
/content/journal/micro/10.1099/mic.0.001455
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