One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy.
One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy.
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DOI：
10.1093/nar/gkt915
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发表时间：
2014-01
影响因子：
14.9
通讯作者：
Ellis T
中科院分区：
文献类型：
--
作者：
Casini A;MacDonald JT;De Jonghe J;Christodoulou G;Freemont PS;Baldwin GS;Ellis T
重叠定向DNA组装方法允许多个DNA部分在一个反应中组装在一起。这些方法依赖于DNA部分末端之间的序列同源性，尽管与工程的一个关键原则：模块化不相容，但已在合成生物学中得到广泛采用。为了回答这个问题，我们提出了MODAL：一种带有连接子的模块化重叠定向组装策略，它为DNA定向方法带来了模块化，允许在一锅反应中将初始的一组DNA部分组装成各种排列。MODAL附带一个定制的软件工具，该工具设计重叠连接器来引导装配，允许零件以任何指定的顺序和方向装配。与使用非设计的序列相比，合成正交重叠接头的计算机设计允许对于各种不同的方法在DNA组装中具有更高的效率。在使用三种不同组装技术的测试中，MODAL策略提供了酵母和细菌质粒的组装，这些质粒由酶范围内的多达五个DNA部分组成，效率在75%至100%之间。它还无缝地允许在该过程中对任何指定的DNA部分进行诱变，从而允许一步创建对合成生物学应用有价值的构建体文库。
Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications.
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