1887

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.

Funding
This study was supported by the:
  • National Health and Medical Research Council (Award GNT1145075)
    • Principle Award Recipient: TimothyP Stinear
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2021-01-07
2021-08-02
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