An improved single cell ultrahigh throughput screening method based on in vitro compartmentalization.
An improved single cell ultrahigh throughput screening method based on in vitro compartmentalization.
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一种改进的基于体外区室化的单细胞超高通量筛选方法
DOI：
10.1371/journal.pone.0089785
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Yang G
中科院分区：
文献类型：
--
作者：
Ma F;Xie Y;Huang C;Feng Y;Yang G
高通量筛选是酶发现和工程的关键技术。基于体外区室化的荧光激活细胞分选（IVC-FACS）最近已成为超高通量生物催化剂筛选的强大工具。然而，当前 IVC-FACS 测定的准确性受到均质产生的微反应器的广泛多分散性的严重限制。在这里，报道了一种基于膜挤出技术的改进方案，以更均匀的方式生成微反应器。这一关键的改进使得能够以> 108个克隆/天的速度对酶活性进行超高通量筛选，其准确度可以区分微反应器内酶活性低至两倍的差异，这比迄今为止报道的类似IVC-FACS系统要高。与本体反应系统相比，微反应器中的酶促反应具有非常相似的动力学行为，并且显示出较宽的动态范围。通过使用改良的 IVC-FACS，通过一轮分选，具有酯酶活性的大肠杆菌细胞可以从大量过量的背景细胞中富集 330 倍。嗜热酯酶 AFEST 的定向进化进一步说明了这种新的 IVC-FACS 系统的实用性。这种非常高效的酯酶的催化活性进一步提高了∼2倍，产生了几种改进的突变体，其k cat/K M 值接近∼108 M−1s−1的扩散限制效率。
High-throughput screening is a key technique in discovery and engineering of enzymes. In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS) has recently emerged as a powerful tool for ultrahigh-throughput screening of biocatalysts. However, the accuracy of current IVC-FACS assays is severely limited by the wide polydispersity of micro-reactors generated by homogenizing. Here, an improved protocol based on membrane-extrusion technique was reported to generate the micro-reactors in a more uniform manner. This crucial improvement enables ultrahigh-throughput screening of enzymatic activity at a speed of >108 clones/day with an accuracy that could discriminate as low as two-fold differences in enzymatic activity inside the micro-reactors, which is higher than similar IVC-FACS systems ever have reported. The enzymatic reaction in the micro-reactors has very similar kinetic behavior compared to the bulk reaction system and shows wide dynamic range. By using the modified IVC-FACS, E. coli cells with esterase activity could be enriched 330-fold from large excesses of background cells through a single round of sorting. The utility of this new IVC-FACS system was further illustrated by the directed evolution of thermophilic esterase AFEST. The catalytic activity of the very efficient esterase was further improved by ∼2-fold, resulting in several improved mutants with k cat/K M values approaching the diffusion-limited efficiency of ∼108 M−1s−1.
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影响因子：
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作者：
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