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

4 Citations (Scopus)


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 Inc.
Number of pages11
ISBN (Electronic)9781071604670
ISBN (Print)9781071604663
Publication statusPublished - 2020

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


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