Development of a rapid method for site-directed mutagenesis in Streptococcus zooepidemicus

Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11110%2F20%3A10413885" target="_blank" >RIV/00216208:11110/20:10413885 - isvavai.cz</a>
Result on the web
<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=95d9iWnN.w" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=95d9iWnN.w</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1016/J.BTECX.2020.100025" target="_blank" >10.1016/J.BTECX.2020.100025</a>

Result language
angličtina
Original language name
Development of a rapid method for site-directed mutagenesis in Streptococcus zooepidemicus
Original language description
This paper describes the development of a straightforward method for site-directed gene mutagenesis in Streptococcus zooepidemicus, inspired by the mechanism of natural competence regulated by ComX in other streptococci. An alternative sigma factor comX gene was overexpressed from a plasmid in S. zooepidemicus and electrocompetent cells were prepared. As proof of concept, a DNA cassette with two targeting regions flanking a kanamycin resistance gene was spliced in an overlap extension PCR and electroporated. The cassette was then integrated in the genomic DNA by homologous recombination. Next, the gene SeseC_00180 (fibrinogen- and Ig-binding protein precursor) was selected as target for markerless gene deletion and the impact of its loss on the resulting hyaluronan production was determined. The new method of site-directed mutagenesis is significant because it is not necessary to clone the DNA cassette in an auxiliary vector, electroporating it in S. zooepidemicus cells is enough, which allows to bypass the problems with hard to clone DNA sequences and speeds up the whole process of mutation generation in S. zooepidemicus.
Czech name
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Czech description
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Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10606 - Microbiology

Publication year
2020
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

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