Engler Carola, Marillonnet Sylvestre
Icon Genetics GmbH, Biozentrum Halle, Halle, Germany.
Methods Mol Biol. 2011;729:167-81. doi: 10.1007/978-1-61779-065-2_11.
Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction-ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction-ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction-ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants.
当前基于限制性内切酶和连接酶使用的标准克隆方法用途广泛，但不太适合高通量克隆项目，也不适合在一步中从几个亲本质粒组装多个DNA片段。我们之前报道了一种基于IIs型限制性内切酶和限制连接的高效克隆方法。这种方法能够高效地无缝组装来自几个亲本质粒的多个片段，并且如果将从几个同源基因制备的片段在一次限制连接中组装在一起，还能进行DNA改组。这种方案称为金门改组，需要执行以下步骤：（1）比对几个同源基因的序列，并在保守序列上定义重组位点；（2）由这些重组位点的位置定义的模块通过PCR扩增，所用引物设计为使其带有侧翼BsaI位点；（3）将扩增的片段作为中间构建体克隆并测序；（4）最后，将中间模块在一个兼容的受体载体中通过一锅法限制连接组装在一起。根据用户需求，并且由于克隆效率高，所得构建体既可以单独筛选和分析，或者如果需要大量构建体，也可以直接用于功能筛选以检测改良的蛋白质变体。
