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

Yuri Ota, Kanako Saito, Taeko Takagi, Satoko Matsukura, Masamune Morita, Satoshi Tsuneda, Naohiro Noda

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Article numbere0214533
JournalPloS one
Volume14
Issue number4
DOIs
Publication statusPublished - 2019 Apr 1

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Nucleic Acid Probes
High-Throughput Screening Assays
RNA Probes
Fluorescence Resonance Energy Transfer
Sorting
sorting
nucleic acids
droplets
Transfer RNA
Bacteria
Screening
Throughput
screening
bacteria
Growth
RNA probes
energy transfer
fluorescence
Fluorescence
methodology

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

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.

In: PloS one, Vol. 14, No. 4, e0214533, 01.04.2019.

Research output: Contribution to journalArticle

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. In: PloS one. 2019 ; Vol. 14, No. 4.
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