Golden Gate Assembly Breakthrough
Golden Gate Assembly Breakthrough
复制标题
金门组装突破
DOI：
10.1089/gen.38.15.05
复制
发表时间：
2018
影响因子：
--
通讯作者：
E. Cantor
中科院分区：
文献类型：
--
作者：
R. Kucera;E. Cantor
Rebecca Kucera 和 Eric Cantor 博士受到合成生物学界的欢迎，Golden Gate 组装通常用于在单个“一锅”反应中组装 2-10 个 DNA 片段，形成复杂的多插入模块化组件，从而实现生物合成途径工程和优化。然而，当前组装超过 10 个模块的最佳实践通常依赖于两步分层方法，在每个步骤中使用不同类型的 IIS 限制性内切酶特异性。酶效率、稳定性和缓冲液兼容性等因素对单步或两步组装产生了实际限制。我们通过使用工程化的 IIS 限制性内切酶并根据实验得出的 DNA 连接酶保真度数据仔细选择功能序列，减少了这些限制。我们的工作表明，现在可以以稳健的效率实现 20 多个片段组装
Rebecca Kucera and Eric Cantor, Ph. D. mbraced by the synthetic biology community, Golden Gate assembly is commonly used to assemble 2–10 DNA fragments in a single “one pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic pathway engineering and optimization. However, current best practices for assemblies of more than 10 modules often rely on two-step hierarchical approaches using different type IIS restriction enzyme specificities at each step. Factors such as enzyme efficiency, stability, and buffer compatibility have placed practical limits on single-or two-step assemblies.We have reduced these limitations through the use of engineered type IIS restriction enzymes and careful choice of function sequences guided by experimentally derived DNA ligase fidelity data. Our work demonstrates that it is now possible to achieve 20-plus fragment assemblies with both robust efficiency