Andreou Andreas I, Nakayama Naomi
SynthSys Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, United Kingdom.
Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, United Kingdom.
PLoS One. 2018 Jan 2;13(1):e0189892. doi: 10.1371/journal.pone.0189892. eCollection 2018.
Synthetic biology builds upon the foundation of engineering principles, prompting innovation and improvement in biotechnology via a design-build-test-learn cycle. A community-wide standard in DNA assembly would enable bio-molecular engineering at the levels of predictivity and universality in design and construction that are comparable to other engineering fields. Golden Gate Assembly technology, with its robust capability to unidirectionally assemble numerous DNA fragments in a one-tube reaction, has the potential to deliver a universal standard framework for DNA assembly. While current Golden Gate Assembly frameworks (e.g. MoClo and Golden Braid) render either high cloning capacity or vector toolkit simplicity, the technology can be made more versatile-simple, streamlined, and cost/labor-efficient, without compromising capacity. Here we report the development of a new Golden Gate Assembly framework named Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. It is based on a two-level, hierarchical approach and utilizes a low-frequency cutter to reduce domestication requirements. Mobius Assembly embraces the standard overhang designs designated by MoClo, Golden Braid, and Phytobricks and is largely compatible with already available Golden Gate part libraries. In addition, dropout cassettes encoding chromogenic proteins were implemented for cost-free visible cloning screening that color-code different cloning levels. As proofs of concept, we have successfully assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements. Taken together, Mobius Assembly delivers enhanced versatility and efficiency in DNA assembly, facilitating improved standardization and automation.
合成生物学建立在工程原理的基础之上，通过设计-构建-测试-学习循环推动生物技术的创新与改进。DNA组装的全社区标准将使生物分子工程在设计和构建的可预测性和通用性水平上达到与其他工程领域相当的程度。金门组装技术具有在单管反应中单向组装大量DNA片段的强大能力，有潜力为DNA组装提供一个通用的标准框架。虽然目前的金门组装框架（如MoClo和Golden Braid）要么具有高克隆能力，要么载体工具包简单，但该技术可以在不影响能力的情况下变得更加通用——简单、简化且成本/劳动力高效。在此，我们报告了一种名为莫比乌斯组装的新型金门组装框架的开发，它将载体工具包的简单性与高克隆能力结合在一起。它基于两级分层方法，并利用低频切割器来降低驯化要求。莫比乌斯组装采用了MoClo、Golden Braid和植物基因砖指定的标准突出端设计，并且在很大程度上与现有的金门部件库兼容。此外，还实施了编码显色蛋白的缺失盒，用于免费的可见克隆筛选，对不同的克隆水平进行颜色编码。作为概念验证，我们已经成功地以操纵子和多基因排列方式组装了多达16个不同色素沉着基因的转录单元。综上所述，莫比乌斯组装在DNA组装中提供了更高的通用性和效率，有助于改进标准化和自动化。
