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Terekhov, Stanislav S.; Smirnov, Ivan V.; Stepanova, Anastasiya V.; Bobik, Tatyana V.; Mokrushina, Yuliana A.; Ponomarenko, Natalia A.; Belogurov, Alexey A.; Rubtsova, Maria P.; Kartseva, Olga V.; Gomzikova, Marina O.; Moskovtsev, Alexey A.; Bukatin, Anton S.; Dubina, Michael V.; Kostryukova, Elena S.; Babenko, Vladislav V.; Vakhitova, Maria T.; Manolov, Alexander I.; Malakhova, Maja V.; Kornienko, Maria A.; Tyakht, Alexander V.; Vanyushkina, Anna A.; Ilina, Elena N.; Masson, Patrick; Gabibov, Alexander G.; Altman, Sidney
Proceedings of the National Academy of Sciences - PNAS, 03/2017, Letnik: 114, Številka: 10Journal Article
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.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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in: SICRIS
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