Directed evolution by in vitro compartmentalization
Directed evolution by in vitro compartmentalization
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DOI：
10.1038/nmeth897
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发表时间：
2006-07-01
期刊：
影响因子：
48
通讯作者：
Griffiths, Andrew D.
中科院分区：
文献类型：
--
作者：
Miller, Oliver J.;Bernath, Kalia;Griffiths, Andrew D.
体外区室化（IVC）的目标是在许多微观区室之间划分大的反应(1)。这项技术最初是为了产生用于蛋白质定向进化的“人造细胞”而开发的（图1）。通常情况下，将基因的水溶液和体外转录-翻译系统搅拌（或均质）到油-表面活性剂混合物中，以产生油包水（w/o）乳液，每毫升乳液中含有10（10）个水滴。大多数液滴只含有一个基因，以及表达该基因所需的所有分子机制。表达的蛋白及其催化活性产物不能离开液滴，因此基因型在体外与表型耦合，使得选择非常大的基因文库(10（8)-10(11)）成为可能。我们在方框1中描述了IVC的优点和应用。在这里，我们提出了一个协议，执行一个定向演化实验的IVC，使一个或多个w/o乳剂的使用。该程序包括基因文库的生成，选择的执行，以及随后通过PCR恢复所选基因。我们还描述了将w/o乳剂转化为水包油包水（w/o/w）乳剂的两种程序，以便使用荧光激活细胞分选器（FACS；框2和图2以及框3）进行高通量筛选。最后，我们描述了两种将底物、调节剂和其他化合物输送到w/o乳液的预成型水滴中的方法（框4）。
The goal of in vitro compartmentalization (IVC) is to divide a large reaction between many microscopic compartments(1). This technique was first developed to generate 'artificial cells' for the directed evolution of proteins (Fig. 1). Typically, an aqueous solution of genes and an in vitro transcription-translation system is stirred (or homogenized) into an oil-surfactant mixture to create a water-in-oil (w/o) emulsion with similar to 10(10) aqueous droplets per ml of emulsion. The majority of droplets contain no more than a single gene along with all of the molecular machinery needed to express that gene. The expressed proteins and the products of their catalytic activities cannot leave the droplets, and so genotype is coupled to phenotype in vitro, making it possible to select very large libraries of genes (10(8)-10(11) genes). We describe the advantages and applications of IVC in Box 1. Here we present a protocol for performing a directed evolution experiment by IVC that makes use of one or more w/o emulsions. This procedure involves the generation of a gene library, the performance of a selection, and the subsequent recovery of the selected genes by PCR. We also describe two procedures for converting w/o emulsions to water-in-oil-in- water (w/o/w) emulsions for high-throughput screening using a fluorescence-activated cell sorter (FACS; Box 2 and Fig. 2, and Box 3). Finally, we describe two methods for delivering substrates, regulators and other compounds to the preformed aqueous droplets of a w/o emulsion (Box 4).