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Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms
In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detecti...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461810/ https://www.ncbi.nlm.nih.gov/pubmed/33013733 http://dx.doi.org/10.3389/fmicb.2020.01929 |
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author | Lindivat, Mathilde Larsen, Aud Hess-Erga, Ole Kristian Bratbak, Gunnar Hoell, Ingunn Alne |
author_facet | Lindivat, Mathilde Larsen, Aud Hess-Erga, Ole Kristian Bratbak, Gunnar Hoell, Ingunn Alne |
author_sort | Lindivat, Mathilde |
collection | PubMed |
description | In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments. |
format | Online Article Text |
id | pubmed-7461810 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-74618102020-10-01 Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms Lindivat, Mathilde Larsen, Aud Hess-Erga, Ole Kristian Bratbak, Gunnar Hoell, Ingunn Alne Front Microbiol Microbiology In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments. Frontiers Media S.A. 2020-08-18 /pmc/articles/PMC7461810/ /pubmed/33013733 http://dx.doi.org/10.3389/fmicb.2020.01929 Text en Copyright © 2020 Lindivat, Larsen, Hess-Erga, Bratbak and Hoell. http://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 Lindivat, Mathilde Larsen, Aud Hess-Erga, Ole Kristian Bratbak, Gunnar Hoell, Ingunn Alne Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms |
title | Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms |
title_full | Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms |
title_fullStr | Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms |
title_full_unstemmed | Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms |
title_short | Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms |
title_sort | bioorthogonal non-canonical amino acid tagging combined with flow cytometry for determination of activity in aquatic microorganisms |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461810/ https://www.ncbi.nlm.nih.gov/pubmed/33013733 http://dx.doi.org/10.3389/fmicb.2020.01929 |
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