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Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates

We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The F...

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Autores principales: Ota, Yuri, Saito, Kanako, Takagi, Taeko, Matsukura, Satoko, Morita, Masamune, Tsuneda, Satoshi, Noda, Naohiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469844/
https://www.ncbi.nlm.nih.gov/pubmed/30995251
http://dx.doi.org/10.1371/journal.pone.0214533
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author Ota, Yuri
Saito, Kanako
Takagi, Taeko
Matsukura, Satoko
Morita, Masamune
Tsuneda, Satoshi
Noda, Naohiro
author_facet Ota, Yuri
Saito, Kanako
Takagi, Taeko
Matsukura, Satoko
Morita, Masamune
Tsuneda, Satoshi
Noda, Naohiro
author_sort Ota, Yuri
collection PubMed
description We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times.
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spelling pubmed-64698442019-05-03 Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates Ota, Yuri Saito, Kanako Takagi, Taeko Matsukura, Satoko Morita, Masamune Tsuneda, Satoshi Noda, Naohiro PLoS One Research Article We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times. Public Library of Science 2019-04-17 /pmc/articles/PMC6469844/ /pubmed/30995251 http://dx.doi.org/10.1371/journal.pone.0214533 Text en © 2019 Ota et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Ota, Yuri
Saito, Kanako
Takagi, Taeko
Matsukura, Satoko
Morita, Masamune
Tsuneda, Satoshi
Noda, Naohiro
Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates
title Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates
title_full Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates
title_fullStr Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates
title_full_unstemmed Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates
title_short Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates
title_sort fluorescent nucleic acid probe in droplets for bacterial sorting (fnap-sort) as a high-throughput screening method for environmental bacteria with various growth rates
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469844/
https://www.ncbi.nlm.nih.gov/pubmed/30995251
http://dx.doi.org/10.1371/journal.pone.0214533
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