TY - JOUR
T1 - Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity
AU - Terekhov, Stanislav S.
AU - Smirnov, Ivan V.
AU - Stepanova, Anastasiya V.
AU - Bobik, Tatyana V.
AU - Mokrushina, Yuliana A.
AU - Ponomarenko, Natalia A.
AU - Belogurov, Alexey A.
AU - Rubtsova, Maria P.
AU - Kartseva, Olga V.
AU - Gomzikova, Marina O.
AU - Moskovtsev, Alexey A.
AU - Bukatin, Anton S.
AU - Dubina, Michael V.
AU - Kostryukova, Elena S.
AU - Babenko, Vladislav V.
AU - Vakhitova, Maria T.
AU - Manolov, Alexander I.
AU - Malakhova, Maja V.
AU - Kornienko, Maria A.
AU - Tyakht, Alexander V.
AU - Vanyushkina, Anna A.
AU - Ilina, Elena N.
AU - Masson, Patrick
AU - Gabibov, Alexander G.
AU - Altman, Sidney
N1 - Publisher Copyright:
© 2017, National Academy of Sciences. All rights reserved.
PY - 2017/3/7
Y1 - 2017/3/7
N2 - Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
AB - Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
KW - Butyrylcholinesterase
KW - Cell-cell interactions
KW - Microfluidic encapsulation
KW - Staphylococcus aureus
KW - Ultrahigh-throughput screening
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UR - http://www.scopus.com/inward/citedby.url?scp=85014656468&partnerID=8YFLogxK
U2 - 10.1073/pnas.1621226114
DO - 10.1073/pnas.1621226114
M3 - Article
C2 - 28202731
AN - SCOPUS:85014656468
SN - 0027-8424
VL - 114
SP - 2550
EP - 2555
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 10
ER -