Protocol

Sorting of water-in-oil emulsions with bacteria using a fluorescent nucleic acid probe

Naohiro Noda, AIST, Biomedical Research Institute

R&D Team, PHC Corporation of North America (PHCNA)

Aug 25, 2021

Abstract

To isolate water-in-oil emulsion droplets containing bacteria from those that do not contain bacteria we have developed a simple and reliable selective sorting method that allows bacteria-containing droplets to be isolated on our microfluidic chip cell sorter, On-chip Sort. Bacteria-containing droplets are sorted based on the fluorescence produced as a result of the interaction between bacteria and fluorescence resonance energy transfer (FRET)-based RNA probe encapsulated inside the droplets. This method is named Fluorescent Nucleic Acid Probe in droplets for Sorting bacteria (FNAP-sort)


Introduction

Uniformly sized water-in-oil emulsion droplets generated in a highly efficient manner by droplet microfluidics-based systems allow each droplet to act as reactors. These small volume reactors have been used in applications such as the cultivation of bacteria, droplet digital PCR, high-throughput screening, and analysis of enzymatic kinetics. In particular, encapsulating a single bacterium into individual droplets prevents the competition between fast- and slow-growing species. This is useful for cultivating rare and slow-growing bacteria separately from the bacterial mixture that contains a vast amount of fast-growing bacteria. However, after the process of encapsulating bacteria into droplets, isolating droplets containing bacteria from those that do not contain bacteria remains technically challenging, resulting in only a very limited number of successful cultivation of environmental microorganisms in the literature. To overcome this challenge, we developed a simple and reliable selective sorting method that allows bacteria-containing droplets to be isolated on our microfluidic chip cell sorter, On-chip Sort.


Procedure

  1. A single Escherichia coli bacterium or bacteria suspended in culture medium with a FRET-based RNA probe was encapsulated in 1-nL water-in-oil emulsion droplets (Figure 1A). 
  2. Droplets were then transferred to a 1.5 mL tube to allow the encapsulated bacterial cells to grow at 37oC for 1 day (Figure 1B).
  3. During incubation, the FRET-based RNA probe does not emit fluorescence by itself; however, in the presence of bacteria, ribonuclease (RNase) secreted by the growing bacteria cleaves the FRET-based RNA probe to generate strong fluorescence inside the droplets (Figure 1C). 
  4. On-chip Sort with microfluidic chip with 150 µm channel (2D-chip Z1000-w150) was used to isolate the highly fluorescent droplets containing growing bacteria (Figure 1D).

Droplets containing E. coli and FRET-based RNA probe were observed to emit weak fluorescence immediately after being generated (Fig. 2a, b). Droplets containing E. coli and FRET-based RNA probe were observed to be brighter after 1 day of incubation at 37 Deg C (Fig. 2 c, d). These results show that the FRET-based RNA probe, when encapsulated with growing bacteria inside the droplets, successfully generated fluorescence observable by dark-field and fluorescence microscopy. Strong fluorescing droplets containing growing E. coli after 1 day of incubation were successfully sorted by On-chip Sort (Fig. 2e, f). Therefore, FNAP-sort, the combined use of FRET-based RNA probe for generation of strong fluorescence upon RNA cleavage by RNAses secreted from growing bacteria inside droplets and subsequently sorting the fluorescent droplets with On-chip Sort, could potentially be a useful tool for high-throughput cultivation and isolation of environmental samples in water-in-oil emulsion droplets. 


Time Taken

1.5 days

Notes and Comments

Under the license of Attribution 4.0 International (CC BY 4.0) https://creativecommons.org/licenses/by/4.0/). 


References

[1] Ota, Y., Saito, K. et al. PLoS ONE 14(4): e0214533. 


Acknowledgments

On-Chip Biotechnologies Co., Ltd. 203 Venture Port, 2-24-16 Naka-cho, Koganei-city, Tokyo 184-0012, Japan TEL: +81-42-385-0461 Email: info@on-chipbio.com URL: https://on-chipbio.com


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