Engler Carola, Gruetzner Ramona, Kandzia Romy, Marillonnet Sylvestre
Icon Genetics GmbH, Biozentrum Halle, Halle, Germany.
PLoS One. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553. Epub 2009 May 14.
We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call 'Golden Gate shuffling', is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
我们开发了一种方法，可在一步操作和一个试管中，将至少九个独立的DNA片段一起组装到一个受体载体中，所获得的重组克隆中有90%包含所需构建体。该方法基于IIs型限制性内切酶的使用，只需将10个未消化的输入质粒（九个插入质粒和受体载体）混合，进行限制性内切酶连接反应，然后将所得混合物转化到感受态细胞中即可。该方法的效率使得通过在一个反应中组合从不同亲本模板制备的几个片段集来生成重组基因文库成为可能。例如，我们已将此策略应用于来自三个亲本模板（牛阳离子胰蛋白酶原、牛阴离子胰蛋白酶原和人阳离子胰蛋白酶原）的胰蛋白酶原改组，每个亲本模板又分为9个独立模块。我们表明，使用27个胰蛋白酶原输入质粒进行一轮改组，在一次限制性内切酶连接反应中就能轻松产生19,683种不同的可能组合，并且对文库的一个子集进行表达筛选可以鉴定出能够导致胰蛋白酶活性更高表达水平的变体。我们将此方法称为“金门改组”，它稳健、简单且高效，可以用无同源性的模板进行操作，并且可以与其他改组方法相结合，以便在给定基因的任何部分引入任何变异。
