Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution
Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution
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DOI：
10.1021/acssynbio.9b00103
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发表时间：
2019-06-01
影响因子：
4.7
通讯作者：
Chaput, John C.
中科院分区：
文献类型：
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作者：
Vallejo, Derek;Nikoomanzar, Ali;Chaput, John C.
合成生物学旨在通过重新利用生物酶用于实际应用来改善人类健康和环境。然而，天然酶在工程化到生物途径中或挑战识别非天然底物时通常以次优活性起作用。克服这个问题需要有效的定向进化方法来发现具有所需活性的新酶变体。在这里，我们描述的建设，验证和应用的荧光激活液滴分选（FADS）仪器，建立了进化酶的合成和修改人工遗传聚合物（XNAs）。微流体系统能够使用响应于酶活性的荧光传感器在类似于2-3 kHz下进行液滴分选。进化出具有定制特性的核酸酶的能力将独特地推动合成生物学、生物技术和医疗保健领域的新兴应用。
Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at similar to 2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.