Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution

Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcomin...

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Bibliographic Details
Published inACS synthetic biology Vol. 8; no. 6; pp. 1430 - 1440
Main Authors Vallejo, Derek, Nikoomanzar, Ali, Paegel, Brian M, Chaput, John C
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 21.06.2019
Subjects
droplet microfluidics
droplet sorting
enzyme engineering
high throughput screening
DrOPS
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Summary:Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2–3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.
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These authors contributed equally to the work
D.V., A.N., and J.C. conceived of the project and designed the experiments. B.P. consulted on optical instrumentation engineering. D.V. and A.N. performed all of the experiments. D.V., A.N., B.P. and J.C. wrote the manuscript. All authors reviewed and commented on the manuscript.
Author Contributions
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.9b00103