European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
Methods Mol Biol. 2025;2850:387-416. doi: 10.1007/978-1-0716-4220-7_22.
Both regulatory sequences and genome organization contribute to the production of diverse transcript isoforms, which can influence how genes, or sets of genes, are expressed. An efficient, modular approach is needed to generate the combinatorial complexity required to empirically test many combinations of different regulatory sequences and different gene orders. Golden Gate assembly provides such a tool for seamless one-pot cleavage and ligation, by using type IIS restriction enzymes, which cleave outside of their recognition site. In addition to reducing the number of steps, this one-pot reaction can improve correct assemblies by the continued cleavage of self-ligation products that retain the recognition site. Switching the specific restriction enzyme used between steps allows for modular assembly of several units. A protocol to perform modular assemblies with two type IIS restriction enzymes, namely BsaI-v2-HF and BsmBI-v2, is described here. This protocol includes a description for generating destination vectors that add loxPsym sites between transcriptional units, allowing for diversification of gene order, orientation, and spacing.
调控序列和基因组组织都有助于产生多样化的转录本异构体，从而影响基因或基因集的表达方式。需要一种高效的模块化方法来产生组合复杂性，以经验性测试不同调控序列和不同基因顺序的许多组合。Golden Gate 组装通过使用 II 型限制酶提供了这样一种工具，这些酶在其识别位点之外切割。除了减少步骤数之外，这种一锅反应还可以通过继续切割保留识别位点的自连接产物来提高正确组装的效率。在步骤之间切换使用的特定限制酶允许几个单元的模块化组装。这里描述了使用两种 II 型限制酶（BsaI-v2-HF 和 BsmBI-v2）进行模块化组装的方案。该方案包括了在转录单元之间生成添加 loxPsym 位点的目的载体的描述，从而可以多样化基因顺序、方向和间距。
