Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
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DOI：
10.1126/sciadv.aav8185
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发表时间：
2019-08
期刊：
影响因子：
13.6
通讯作者：
Yi Zhang;Yoshihiro Minagawa;H. Kizoe;K. Miyazaki;R. Iino;H. Ueno;K. Tabata;Y. Shimane;H. Noji
中科院分区：
文献类型：
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作者：
Yi Zhang;Yoshihiro Minagawa;H. Kizoe;K. Miyazaki;R. Iino;H. Ueno;K. Tabata;Y. Shimane;H. Noji
基于泊松统计的策略使得能够从仅几个克隆中快速有效地获得酶突变体。我们报告了一个通用的战略数字计数原理的基础上，使一个有效的收购酶突变体所需的活动，从几个克隆在一天之内。我们制备了一个高密度的毫微微升液滴阵列，每平方厘米由100万个均匀的液滴组成，以进行高通量蛋白质合成和筛选。单个DNA分子随机分布到每个液滴中，遵循泊松过程，以启动蛋白质合成，伴随无细胞转录和翻译反应，然后通过微毛细管回收。每个液滴中的蛋白质产量与DNA分子的数量成比例，这意味着具有高于泊松分布预测的最大值的表观强度的液滴可以容易地被鉴定为表现出期望的增加的活性的精确命中。我们通过使用少于10 nl的试剂将碱性磷酸酶的活性提高了近20倍。
A strategy based on Poisson statistics enables a rapid and efficient acquisition of enzyme mutants from just a few clones. 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.