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

Kód výsledku v 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>

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

—

Návaznosti

V - Vyzkumna aktivita podporovana z jinych verejnych zdroju

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Biotechnology

ISSN

0168-1656

e-ISSN

—

Svazek periodika

324

Číslo periodika v rámci svazku

Suppl.

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

9

Strana od-do

100025

Kód UT WoS článku

000710701700010

EID výsledku v databázi Scopus

2-s2.0-85090027239