A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution
A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution
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DOI：
10.1039/c2lc21035e
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发表时间：
2012-01-01
期刊：
影响因子：
6.1
通讯作者：
Griffiths, Andrew D.
中科院分区：
文献类型：
--
作者：
Fallah-Araghi, Ali;Baret, Jean-Christophe;Griffiths, Andrew D.
体外筛选系统的基础上耦合的转录和翻译的基因使用无细胞系统具有许多有吸引力的功能，蛋白质工程和定向进化。我们提出了一个完全在体外超高通量筛选平台，使用液滴为基础的微流体。将单个基因区室化在水性液滴中，分散在惰性载体油中，并使用聚合酶链反应（PCR）扩增。扩增后，将现在含有每个基因的30000个拷贝的液滴与含有无细胞偶联转录-翻译（IVTT）系统和用于荧光测定的试剂的液滴一对一地融合。然后使用与水流的荧光激活电聚结来选择性地从含有所需活性的液滴中回收基因。通过选择编码β-半乳糖苷酶的lacZ基因和编码非活性变体的lacZmut基因的混合物，我们证明了该系统可以在2000个液滴s（-1）下进行分选：lacZ基因从1：100的lacZ：lacZmut基因摩尔比中富集了502倍。实际上，假阳性和假阴性错误率均< 0.004，并且结果表明富集不受分选效率的限制，而是受液滴中多个基因的共包封的限制，这由泊松分布描述。与使用基于微量滴定板的系统进行筛选相比，PCR和IVTT试剂的体积和成本降低了近10（5）倍，允许仅使用150 μ L试剂筛选10（6）个基因。
In vitro screening systems based on the coupled transcription and translation of genes using cell-free systems have a number of attractive features for protein engineering and directed evolution. We present a completely in vitro ultrahigh-throughput screening platform using droplet-based microfluidics. Single genes are compartmentalized in aqueous droplets, dispersed in inert carrier oil, and amplified using the polymerase chain reaction (PCR). After amplification, the droplets, now containing 30 000 copies of each gene, are fused one-to-one with droplets containing a cell-free coupled transcription-translation (IVTT) system and the reagents for a fluorogenic assay. Fluorescence-activated electrocoalescence with an aqueous stream is then used to selectively recover genes from droplets containing the desired activity. We demonstrate, by selecting mixtures of lacZ genes encoding the enzyme beta-galactosidase and lacZmut genes encoding an inactive variant, that this system can sort at 2000 droplets s(-1): lacZ genes were enriched 502-fold from a 1 : 100 molar ratio of lacZ : lacZmut genes. Indeed, the false positive and false negative error rates were both < 0.004 and the results indicate that enrichment is not limited by the sorting efficiency, but by the co-encapsulation of multiple genes in droplets, which is described by the Poisson distribution. Compared to screening using microtiter plate-based systems, the volume and cost of PCR and IVTT reagents are reduced by almost 10(5)-fold, allowing the screening of 10(6) genes using only 150 mu L of reagents.