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Volume 3, Issue 2

From cloning to mutant in 5 days: rapid allelic exchange in Open Access

Abstract

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in and should dramatically improve the scalability of gene-function studies.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): allelic exchange , Electroporation , mutagenesis , Staphylococcus aureus , temperature sensitive and Transformation
Funding
This study was supported by the:
  • National Health and Medical Research Council (Award GNT1145075)
    • Principle Award Recipient: TimothyP Stinear
© Crown copyright 2021
  • This information is licensed under the Open Government Licence 3.0. This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2021-01-07
2024-05-10
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From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus
Access Microbiology 3, 000193 (2020); https://doi.org/10.1099/acmi.0.000193
/content/journal/acmi/10.1099/acmi.0.000193
/content/journal/acmi/10.1099/acmi.0.000193
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