Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly.
Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly.
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DOI：
10.1371/journal.pone.0189892
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Nakayama N
中科院分区：
文献类型：
--
作者：
Andreou AI;Nakayama N
合成生物学建立在工程原理的基础上，通过设计-构建-测试-学习循环促进生物技术的创新和改进。在DNA组装方面的全社区标准将使生物分子工程在设计和建造方面具有可与其他工程领域相媲美的预测性和普遍性。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，有可能为DNA组装提供通用的标准框架。虽然目前的Golden Gate Assembly框架（例如MoClo和Golden Braid）提供了高克隆能力或载体工具包简单性，但该技术可以变得更加简单，简化和成本/劳动效率高，而不会影响容量。在这里，我们报告了一个名为Mobius Assembly的新的Golden Gate Assembly框架的开发，它结合了向量工具包的简单性和高克隆能力。它基于两级分层方法，并利用低频切割机来降低驯化要求。Mobius Assembly采用MoClo、Golden Braid和Phytobricks指定的标准悬挑设计，并与现有的Golden Gate零件库基本兼容。此外，实施了编码显色蛋白的dropout盒，用于对不同克隆水平进行颜色编码的免费可见克隆筛选。作为概念的证据，我们已经成功地组装了多达16个转录单位的各种色素基因的操纵子和多基因的安排。总之，Mobius Assembly在DNA组装中提供了增强的多功能性和效率，促进了标准化和自动化的改进。
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.
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