New applications of pHluorin - measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11320%2F10%3A10062282" target="_blank" >RIV/00216208:11320/10:10062282 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/67985823:_____/10:00345607

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

New applications of pHluorin - measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Popis výsledku v původním jazyce

pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pHin) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pHin for three prototrophic wild-types. The new construct was also used to determine the pHin in strains differing in the activity of the plasma membrane Pma1 H+-ATPase and the influence of glucose on pHin. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We alsoutilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca.52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

Název v anglickém jazyce

New applications of pHluorin - measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Popis výsledku anglicky

pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pHin) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pHin for three prototrophic wild-types. The new construct was also used to determine the pHin in strains differing in the activity of the plasma membrane Pma1 H+-ATPase and the influence of glucose on pHin. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We alsoutilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca.52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

BO - Biofyzika

OECD FORD obor

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Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>Z - Vyzkumny zamer (s odkazem do CEZ)

Rok uplatnění

2010

Kód důvěrnosti údajů

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

Název periodika

Yeast

ISSN

0749-503X

e-ISSN

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Svazek periodika

27

Číslo periodika v rámci svazku

6

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

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Kód UT WoS článku

000279060200002

EID výsledku v databázi Scopus

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