2020
DOI: 10.1099/acmi.0.000193
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From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus

Abstract: 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 exc… Show more

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Cited by 29 publications
(23 citation statements)
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“…To construct the double deletion of stp / pknB and introduce the noncatalytic pknB (K39M) mutation, we followed the protocol of Monk and Stinear ( 77 ). Genomic DNA from either USA300 or Cowan I was used as a template for the PCR amplification (primers are described in table S3) of the respective constructs by splicing by overhang extension (SOE)-PCR.…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…To construct the double deletion of stp / pknB and introduce the noncatalytic pknB (K39M) mutation, we followed the protocol of Monk and Stinear ( 77 ). Genomic DNA from either USA300 or Cowan I was used as a template for the PCR amplification (primers are described in table S3) of the respective constructs by splicing by overhang extension (SOE)-PCR.…”
Section: Methodsmentioning
confidence: 99%
“…Genomic DNA from either USA300 or Cowan I was used as a template for the PCR amplification (primers are described in table S3) of the respective constructs by splicing by overhang extension (SOE)-PCR. Amplimers were introduced into pIMAY-Z by seamless ligation cloning extract (SLiCE) cloning and then transformed into either E. coli IM08B for USA300 or E. coli IM30B for Cowan I. Electroporation into S. aureus , allelic exchange, and conformation by whole-genome sequencing (Illumina, NextSeq) were conducted as described previously ( 77 ). To screen for the PknB K39M point mutation, we performed colony PCR with primers IM1535/IM255 (annealing at 66°C; table S3).…”
Section: Methodsmentioning
confidence: 99%
“…Engineering of convergent mutations in agrA , agrC , ausA, mfd , lysA, and cydA genes in the strain BPH3370 was performed by allelic exchange as described previously (Monk & Stinear, 2021) using oligonucleotides described in supplementary table 7 (outlining residues modified by convergent mutations). Upstream and downstream regions of each mutation were PCR amplified and gel extracted and then a splice by overlap extension (SOE) PCR was performed to generate each insert.…”
Section: Methodsmentioning
confidence: 99%
“…Successful transformants were selected on chloramphenicol (10 μg/ml) plates. The plasmid was isolated using the Monarch plasmid DNA miniprep kit (NEB), sequenced to confirm correct assembly, and transformed into electrocompetent S. xylosus cells as previously described (Monk & Stinear, 2021; Schiffer et al, 2021). Hereby, the transformation of wildtype strains was performed to obtain Δ sxs A mutants, as well as bap ‐deficient mutants (Schiffer et al, 2021) were transformed, to generate Δ bap Δ sxs A mutant strains.…”
Section: Methodsmentioning
confidence: 99%