Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo
Open Access
- 7 August 2020
- journal article
- research article
- Published by American Association for the Advancement of Science (AAAS) in Science Advances
- Vol. 6 (32), eabb3521
- https://doi.org/10.1126/sciadv.abb3521
Abstract
The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min−1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min−1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application.This publication has 40 references indexed in Scilit:
- Advancing oleaginous microorganisms to produce lipid via metabolic engineering technologyProgress in Lipid Research, 2013
- A look at diacylglycerol acyltransferases (DGATs) in algaeJournal of Biotechnology, 2012
- In vivo lipidomics using single-cell Raman spectroscopyProceedings of the National Academy of Sciences of the United States of America, 2011
- Ultrahigh-throughput screening in drop-based microfluidics for directed evolutionProceedings of the National Academy of Sciences of the United States of America, 2010
- Five hard truths for synthetic biologyNature, 2010
- Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activityLab on a Chip, 2009
- Raman tweezers sorting of single microbial cellsEnvironmental Microbiology Reports, 2009
- Production of the antimalarial drug precursor artemisinic acid in engineered yeastNature, 2006
- Schizosaccharomyces pombe Cells Deficient in Triacylglycerols Synthesis Undergo Apoptosis upon Entry into the Stationary PhaseOnline Journal of Public Health Informatics, 2003
- Storage Lipid Synthesis Is Non-essential in YeastJournal of Biological Chemistry, 2002