Optimization of flow cytometry parameters for high-throughput screening of spores of the filamentous fungus Trichoderma reesei.
J Biotechnol
; 321: 78-86, 2020 Sep 10.
Article
em En
| MEDLINE
| ID: mdl-32619643
ABSTRACT
Flow cytometry (FCM) is a powerful technique still little used to study filamentous fungi due to physical constraints the hyphae are too large to enter the FCM fluidic system, unless spores can be analyzed at a very early stage of germination. The technique nevertheless has strong potential for the study of these micro-organisms (spore sorting, viability, characterization etc.). This study focused on the investigation of several parameters, ranging from germination and storage conditions of T. reesei spores through to FCM gating, to detect their fluorescence during the first 24â¯h of germination. Fluorescent spores were first obtained after aerobic germination at 25⯰C and monitored over 24â¯h using FCM, to screen for nine promoters controlling a green fluorescent protein gene. The fluorescence signal (FL1) was then acquired, in addition to the growth characterization of the spores, based on the size signal or Forward Scatter (FSC). They were combined to identify the best candidate(s) from among the nine promoters for the strongest- and earliest-possible fluorescence emission, which resulted in the following ranking pTEFâ¯>â¯pPKIâ¯>â¯pGPDâ¯>â¯pPDC. There are numerous possible applications of this work, ranging from molecular biology to monitoring fermentation.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Esporos Fúngicos
/
Ensaios de Triagem em Larga Escala
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Citometria de Fluxo
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Hypocreales
Tipo de estudo:
Diagnostic_studies
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Screening_studies
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article