Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput.
Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput.
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DOI：
10.1021/acssynbio.0c00538
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发表时间：
2021-02-19
影响因子：
4.7
通讯作者：
Hollfelder F
中科院分区：
文献类型：
--
作者：
Holstein JM;Gylstorff C;Hollfelder F
油包水乳液液滴中单基因的区室化是创建数百万个用于酶库选择的反应器的强大方法。当这些液滴在微流体装置中以超高通量形成时，它们完美的单分散性允许具有高精度读数的定量酶测定。然而，尽管其具有高质量无细胞筛选实验的潜力，但之前的富集演示从未成功地在单分散微流体液滴中进行实际的酶库选择。在这里，我们开发了一个三步工作流程，将三个以前不兼容的步骤分开，迄今为止无法立即执行：首先通过滚环扩增来扩增液滴区室化的 DNA；仅在完成此步骤后，才可使用用于体外蛋白质表达的试剂，最后通过微微注射添加底物。分段工作流程足够强大，可以在液滴中进行首次体外进化，改进对大肠杆菌有毒的蛋白酶 Savinase，使其具有更高的活性，并鉴定出速度快 5 倍的酶。
Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for in vitro protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first in vitro evolution in droplets, improving the protease Savinase that is toxic to E. coli for higher activity and identifying a 5-fold faster enzyme.
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影响因子：
5
作者：
Neveu, Julie;Regeard, Christophe;DuBow, Michael S.
通讯作者：
DuBow, Michael S.
影响因子：
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Colin PY;Kintses B;Gielen F;Miton CM;Fischer G;Mohamed MF;Hyvönen M;Morgavi DP;Janssen DB;Hollfelder F
通讯作者：
Hollfelder F
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通讯作者：
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