Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution
Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution
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DOI：
10.1016/j.chembiol.2012.06.009
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发表时间：
2012-08-24
影响因子：
--
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
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作者：
Kintses, Balint;Hein, Christopher;Hollfelder, Florian
我们展示了微流体平台的实用性，其中油包水液滴隔室用于将细胞裂解物测定法放大一百万倍用于定向酶进化。筛选混杂硫酸酯酶的水解活性表明，这种极端小型化到单细胞水平并不以高信号质量为代价。此外，定量读出提供了以前限于具有有限通量的筛选方法的精确度水平。每轮进化分选3 × 10（7）个单分散液滴导致活性（6倍）和表达（6倍）提高的克隆富集。检测大量筛选克隆中的细微差异提供了拯救停滞的定向进化实验并使其可行所需的高灵敏度和高通量的组合。
We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 x 10(7) monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable.