Ultrahigh-throughput screening in drop-based microfluidics for directed evolution
Ultrahigh-throughput screening in drop-based microfluidics for directed evolution
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DOI：
10.1073/pnas.0910781107
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发表时间：
2010-03-02
影响因子：
11.1
通讯作者：
Weitz, David A.
中科院分区：
文献类型：
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作者：
Agresti, Jeremy J.;Antipov, Eugene;Weitz, David A.
我们对基因组、蛋白质组和代谢组知识的爆炸性增长正在推动对生命生物化学的基本理解不断加深，从而能够对复杂的生物系统及其进化进行定性的新研究。这些知识还推动了分子工程和合成生物学等现代生物技术的发展，这些技术具有解决紧迫问题的巨大潜力，包括开发有效的新药和提供环保能源。然而，其中许多研究最终受到对生化反应更高通量测量的需求的限制。我们提出了一种使用基于液滴的微流体的通用超高通量筛选平台，该平台克服了这些限制并彻底改变了筛选的规模和速度。我们使用分散在油中的水滴作为皮升体积的反应容器，并以每秒数千的速度对其进行筛选。为了证明其威力，我们将该系统应用于定向进化，鉴定出辣根过氧化物酶的新突变体，其催化速率比其亲本快 10 倍以上，而后者已经是一种非常高效的酶。我们利用超高通量进行初始纯化选择，去除不活跃的突变体；我们从类似于 10(7) 的初始群体中识别出大约 100 个活性与亲本相当的变体。经过第二代诱变和高严格筛选，我们鉴定了几个显着改进的突变体，其中一些接近扩散限制效率。总共，我们在短短 10 小时内筛选了类似 10(8) 个单独的酶反应，使用
The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify similar to 100 variants comparable in activity to the parent from an initial population of similar to 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen similar to 10(8) individual enzyme reactions in only 10 h, using