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


ABSTRACT: 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 cm2 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.

SUBMITTER: Zhang Y 

PROVIDER: S-EPMC6703874 | biostudies-other | 2019 Aug

REPOSITORIES: biostudies-other

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

Zhang Yi Y   Minagawa Yoshihiro Y   Kizoe Hiroto H   Miyazaki Kentaro K   Iino Ryota R   Ueno Hiroshi H   Tabata Kazuhito V KV   Shimane Yasuhiro Y   Noji Hiroyuki H  

Science advances 20190821 8


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<sup>2</sup> 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 c  ...[more]