Genetic Manipulation of Group A Streptococcus-Gene Deletion by Allelic Replacement

Timothy C Barnett, Jessica N Daw, Mark J Walker, Stephan Brouwer

Research output: Chapter in Book/Conference paperChapter


Genetic manipulation of Streptococcus pyogenes (Group A Streptococcus, GAS) has historically been a challenging process, with considerable variation in efficiency between different strains. Here, we outline an optimized, rapid method for creating markerless isogenic mutations that combines Gibson assembly cloning with a new temperature-sensitive plasmid, pLZts. This method is highly efficient and reduces the time needed to create GAS mutants to ~2-3 weeks, with the ability to prepare multiple mutants simultaneously.

Original languageEnglish
Title of host publicationGroup A Streptococcus
Subtitle of host publicationMethods and Protocols
EditorsThomas Proft, Jacelyn M. S. Loh
Place of PublicationUSA
PublisherHumana Press
Number of pages11
ISBN (Electronic)9781071604670
ISBN (Print)9781071604663
Publication statusE-pub ahead of print - 20 May 2020

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

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    Improving Health Outcomes in the Tropical North - A Multidisciplinary Collaboration (HOT NORTH)

    Carapetis, J., Currie, B., Anstey, N., McBride, W., Chang, A., Garvey, G., Maple-Brown, L. & Sorrell, T.

    National Health & Medical Research Council NHMRC


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    Cite this

    Barnett, T. C., Daw, J. N., Walker, M. J., & Brouwer, S. (2020). Genetic Manipulation of Group A Streptococcus-Gene Deletion by Allelic Replacement. In T. Proft, & J. M. S. Loh (Eds.), Group A Streptococcus: Methods and Protocols (Vol. 2136, pp. 59-69). (Methods in molecular biology (Clifton, N.J.)). Humana Press.