Abstract
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.
Highlights
Synthetic biology is a fast expanding field at the interface of biology and engineering that facilitates predictive engineering of living organisms with novel functionalities [1]
The Mobius Assembly framework commences at Level 0, which represents the standard part library. It uses the Mobius Universal Acceptor Vector, to convert amplified PCR fragments into standard, exchangeable parts (Fig 1A). mUAV has a backbone derived from pSB1C3 and confers chloramphenicol resistance
We have developed a simple yet versatile new Golden Gate Assembly framework, which was named Mobius Assembly for its unlimited cloning scheme that shifts between two tiers
Summary
Synthetic biology is a fast expanding field at the interface of biology and engineering that facilitates predictive engineering of living organisms with novel functionalities [1]. Engineering principles, such as standardization, modularity, and simplicity, are implemented with the aim of reducing the unpredictability of complex and often non-linear living systems. Standardized DNA parts with consistent and well-characterized functionalities can be utilized just like man-. Org) University Research Fellowship UF140640 to N. N. The University of Edinburgh (www.ed.ac.uk) Chancellor’s Fellowship to N. N. The University of Edinburgh (www.ed.ac.uk) Principal’s Career Development PhD Studentship to A.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
