Zheng Yu, Roberts Richard J
New England BioLabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Res. 2007;35(11):e83. doi: 10.1093/nar/gkm410. Epub 2007 Jun 12.
We describe in this article an in vitro system for the selection of restriction endonucleases using artificial cells. The artificial cells are generated in the form of a water-in-oil emulsion by in vitro compartmentalization. Each aqueous compartment contains a reconstituted transcription/translation mix along with the dispersed DNA templates. In the compartments containing endonuclease genes, an endonuclease expressed in vitro cleaves its own DNA template adjacent to the gene, leaving a sticky end. The pooled DNA templates are then ligated to an adaptor with a compatible end. The endonuclease genes are then enriched by adaptor-specific PCR on the ligation mix. We demonstrate that the system can achieve at least 100-fold enrichment in a single round of selection. It is sensitive enough to enrich an active endonuclease gene from a 1:10(5) model library in 2-3 rounds of selection. Finally, we describe experiments where we selected endonuclease genes directly from a bacterial genomic DNA source in three rounds of selections: the known PstI gene from Providencia stuartii and the new TspMI gene from Thermus sp. manalii. This method provides a unique tool for cloning restriction endonuclease genes and has many other potential applications.
我们在本文中描述了一种利用人工细胞筛选限制性内切酶的体外系统。人工细胞通过体外分隔以油包水乳液的形式产生。每个水相区室都含有重构的转录/翻译混合物以及分散的DNA模板。在含有内切酶基因的区室中，体外表达的内切酶会在基因附近切割其自身的DNA模板，留下粘性末端。然后将汇集的DNA模板与具有兼容末端的接头连接。接着通过连接混合物上的接头特异性PCR对内切酶基因进行富集。我们证明该系统在一轮筛选中可以实现至少100倍的富集。它足够灵敏，能够在2至3轮筛选中从1:10⁵的模型文库中富集到活性内切酶基因。最后，我们描述了在三轮筛选中直接从细菌基因组DNA来源中筛选内切酶基因的实验：来自斯氏普罗威登斯菌的已知PstI基因和来自嗜热栖热放线菌的新TspMI基因。该方法为克隆限制性内切酶基因提供了一种独特的工具，并且还有许多其他潜在应用。
