Kintses Balint, Hein Christopher, Mohamed Mark F, Fischlechner Martin, Courtois Fabienne, Lainé Céline, Hollfelder Florian
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
Chem Biol. 2012 Aug 24;19(8):1001-9. doi: 10.1016/j.chembiol.2012.06.009.
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 × 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.
我们展示了一种微流控平台的实用性，其中油包水液滴隔室用于将细胞裂解物检测微型化一百万倍，以用于定向酶进化。对一种混杂硫酸酯酶的水解活性进行筛选表明，这种极端微型化至单细胞水平并不会在信号质量上付出高昂代价。此外，定量读数提供了一种以前仅限于通量受限的筛选方法的精度水平。每轮进化对3×10⁷个单分散液滴进行分选，导致活性（6倍）和表达（6倍）得到改善的克隆富集。在大量筛选克隆中检测细微差异，提供了挽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。
