From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus.
Ian R MonkTimothy P StinearPublished in: Access microbiology (2021)
In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus 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 Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus 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 S. aureus and should dramatically improve the scalability of gene-function studies.
Keyphrases
- staphylococcus aureus
- escherichia coli
- circulating tumor
- biofilm formation
- randomized controlled trial
- crispr cas
- cell free
- single molecule
- copy number
- methicillin resistant staphylococcus aureus
- cancer therapy
- genome wide
- nucleic acid
- gene expression
- drug delivery
- circulating tumor cells
- pseudomonas aeruginosa
- dna methylation
- cystic fibrosis
- gestational age
- loop mediated isothermal amplification