Generation of Families of Construct Variants Using Golden Gate Shuffling
Generation of Families of Construct Variants Using Golden Gate Shuffling
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DOI：
10.1007/978-1-61779-065-2_11
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Marillonnet, Sylvestre
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文献类型：
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作者：
Engler, Carola;Marillonnet, Sylvestre
目前基于使用限制性酶和连接酶的标准克隆方法是非常通用的，但不太适合高通量克隆项目或在单个步骤中组装来自几个亲本质粒的许多DNA片段。我们以前已经报道了一种有效的克隆方法的基础上使用I型限制性内切酶和限制性连接的发展。这样的方法允许以高效率无缝组装来自几个亲本质粒的多个片段，并且如果从几个同源基因制备的片段在单个限制性连接中组装在一起，则还允许进行DNA改组。这种称为Golden Gate shuffling的方案需要进行以下步骤：（1）将来自几个同源基因的序列进行比对，并在保守序列上确定重组位点;（2）通过PCR扩增由这些重组位点的位置确定的模块，所述PCR使用设计为使它们具有侧翼BsaI位点的引物;（3）将扩增的片段克隆为中间构建体并测序;和（4）最后，在一锅限制性连接中，将中间模块组装在相容的受体载体中。根据用户的需求，并且由于克隆效率高，所得构建体可以单独筛选和分析，或者，如果需要大量，直接用于功能筛选以检测改进的蛋白质变体。
Current standard cloning methods based on the use of restriction enzymes and ligase arc 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 I Is 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.