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Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array

We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm(2) to carry out...

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Autores principales: Zhang, Yi, Minagawa, Yoshihiro, Kizoe, Hiroto, Miyazaki, Kentaro, Iino, Ryota, Ueno, Hiroshi, Tabata, Kazuhito V., Shimane, Yasuhiro, Noji, Hiroyuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703874/
https://www.ncbi.nlm.nih.gov/pubmed/31457078
http://dx.doi.org/10.1126/sciadv.aav8185
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author Zhang, Yi
Minagawa, Yoshihiro
Kizoe, Hiroto
Miyazaki, Kentaro
Iino, Ryota
Ueno, Hiroshi
Tabata, Kazuhito V.
Shimane, Yasuhiro
Noji, Hiroyuki
author_facet Zhang, Yi
Minagawa, Yoshihiro
Kizoe, Hiroto
Miyazaki, Kentaro
Iino, Ryota
Ueno, Hiroshi
Tabata, Kazuhito V.
Shimane, Yasuhiro
Noji, Hiroyuki
author_sort Zhang, Yi
collection PubMed
description We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm(2) to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution–predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.
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spelling pubmed-67038742019-08-27 Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array Zhang, Yi Minagawa, Yoshihiro Kizoe, Hiroto Miyazaki, Kentaro Iino, Ryota Ueno, Hiroshi Tabata, Kazuhito V. Shimane, Yasuhiro Noji, Hiroyuki Sci Adv Research Articles We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm(2) to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution–predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents. American Association for the Advancement of Science 2019-08-21 /pmc/articles/PMC6703874/ /pubmed/31457078 http://dx.doi.org/10.1126/sciadv.aav8185 Text en Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). http://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (http://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Research Articles
Zhang, Yi
Minagawa, Yoshihiro
Kizoe, Hiroto
Miyazaki, Kentaro
Iino, Ryota
Ueno, Hiroshi
Tabata, Kazuhito V.
Shimane, Yasuhiro
Noji, Hiroyuki
Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
title Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
title_full Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
title_fullStr Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
title_full_unstemmed Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
title_short Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
title_sort accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703874/
https://www.ncbi.nlm.nih.gov/pubmed/31457078
http://dx.doi.org/10.1126/sciadv.aav8185
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