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9. Integration of Droplet Microfluidic Tools for Single-Cell Functional Metagenomics: An Engineering Head Start

Author

Conchouso, David, Al-Ma’abadi, Amani, Behzad, Hayedeh, Alarawi, Mohammed, Hosokawa, Masahito, Nishikawa, Yohei, Takeyama, Haruko, Mineta, Katsuhiko, and Gojobori, Takashi

Abstract

Droplet microfluidic techniques have shown promising outcome to study single cellsat high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant due to the complex and multidisciplinary nature of the field. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single cells in droplets at a rate of ∼250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescence-activated droplet sorting systems to integrate the use of 4 independent fluorescence-exciting lasers (i.e., 405, 488, 561, and 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger is also integrated into our pipeline to enable adding new reagents to already-made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput capability (>50,000 cells/day) for mining and bioprospecting metagenomic data

Published
2021
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31. Identification of lipolytic enzymes using high-throughput single-cell screening and sorting of a metagenomic library.

Author

Alma'abadi, Amani, Behzad, Hayedeh, Alarawi, Mohammed, Conchouso, David, Saito, Yoshimoto, Hosokawa, Masahito, Nishikawa, Yohei, Kogawa, Masato, Takeyama, Haruko, Mineta, Katsuhiko, and Gojobori, Takashi

Subjects
*LIPOLYTIC enzymes, *MICROFLUIDIC devices, *HIGH throughput screening (Drug development), *INDUSTRIAL enzymology, *MICROBIAL genes, *METAGENOMICS, *MICROBIAL enzymes, *LIPASES
Abstract

The demand for novel, robust microbial biocatalysts for use in industrial and pharmaceutical applications continues to increase rapidly. As a result, there is a need to develop advanced tools and technologies to exploit the vast metabolic potential of unculturable microorganisms found in various environments. Single-cell and functional metagenomics studies can explore the enzymatic potential of entire microbial communities in a given environment without the need to culture the microorganisms. This approach has contributed substantially to the discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples, constructing expression libraries comprising the entire microbial genome, and screening of the libraries for the presence of desired phenotypes. In this study, lipolytic enzymes from the Red Sea were targeted. A high-throughput single-cell microfluidic platform combined with a laser-based fluorescent screening bioassay was employed to discover new genes encoding lipolytic enzymes. Analysis of the metagenomic library led to the identification of three microbial genes encoding lipases based on their functional similarity and sequence homology to known lipases. The results demonstrated that microfluidics is a robust technology that can be used for screening in functional metagenomics. The results also indicate that the Red Sea is a promising, under-investigated source of new genes and gene products. • Development of a workflow for high-throughput single-cell screening to detect novel enzymes. • Identification of three microbial lipase genes from the metagenome of the Red Sea. • Discovery of microbial enzymes for industrial applications by functional screening. [ABSTRACT FROM AUTHOR]

Published
2022
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