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Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies

Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the p...

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Autores principales: Hartmann, Fabian S. F., Weiß, Tamara, Kastberg, Louise L. B., Workman, Christopher T., Seibold, Gerd M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10303141/
https://www.ncbi.nlm.nih.gov/pubmed/37389345
http://dx.doi.org/10.3389/fmicb.2023.1187228
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author Hartmann, Fabian S. F.
Weiß, Tamara
Kastberg, Louise L. B.
Workman, Christopher T.
Seibold, Gerd M.
author_facet Hartmann, Fabian S. F.
Weiß, Tamara
Kastberg, Louise L. B.
Workman, Christopher T.
Seibold, Gerd M.
author_sort Hartmann, Fabian S. F.
collection PubMed
description Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Toward versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry in Corynebacterium glutamicum, or promoter activity using GFP as reporter in Saccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analyses via imaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA in Escherichia coli colonies. Applicability of this novel technique was further demonstrated by assessing redox states in C. glutamicum colonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology.
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spelling pubmed-103031412023-06-29 Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies Hartmann, Fabian S. F. Weiß, Tamara Kastberg, Louise L. B. Workman, Christopher T. Seibold, Gerd M. Front Microbiol Microbiology Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Toward versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry in Corynebacterium glutamicum, or promoter activity using GFP as reporter in Saccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analyses via imaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA in Escherichia coli colonies. Applicability of this novel technique was further demonstrated by assessing redox states in C. glutamicum colonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology. Frontiers Media S.A. 2023-06-14 /pmc/articles/PMC10303141/ /pubmed/37389345 http://dx.doi.org/10.3389/fmicb.2023.1187228 Text en Copyright © 2023 Hartmann, Weiß, Kastberg, Workman and Seibold. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Hartmann, Fabian S. F.
Weiß, Tamara
Kastberg, Louise L. B.
Workman, Christopher T.
Seibold, Gerd M.
Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
title Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
title_full Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
title_fullStr Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
title_full_unstemmed Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
title_short Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
title_sort precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10303141/
https://www.ncbi.nlm.nih.gov/pubmed/37389345
http://dx.doi.org/10.3389/fmicb.2023.1187228
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