Abstract
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.
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Acknowledgments
The authors would like to thank Dr. Amanda Walker for technical assistance with preparing pLZts mutants. Timothy Barnett is supported by a Career Development Fellowship from the NHMRC-funded Improving Health Outcomes in the Tropical North: A multidisciplinary collaboration (Hot North; APP1131932). Mark Walker is supported by an NHMRC Principal Research Fellowship (APP1102621). The authors acknowledge support from the NHMRC research granting system and the Western Australia Department of Health.
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Barnett, T.C., Daw, J.N., Walker, M.J., Brouwer, S. (2020). Genetic Manipulation of Group A Streptococcus—Gene Deletion by Allelic Replacement. In: Proft, T., Loh, J. (eds) Group A Streptococcus. Methods in Molecular Biology, vol 2136. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0467-0_5
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DOI: https://doi.org/10.1007/978-1-0716-0467-0_5
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