Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
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...
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Published in | PloS one Vol. 13; no. 1; p. e0189892 |
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Main Authors | , |
Format | Journal Article |
Language | English |
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United States
Public Library of Science
02.01.2018
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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. |
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AbstractList | 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. |
Audience | Academic |
Author | Nakayama, Naomi Andreou, Andreas I |
AuthorAffiliation | 3 Centre for Science at Extreme Condition, University of Edinburgh, Edinburgh, United Kingdom 1 SynthSys Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, United Kingdom Imperial College London, UNITED KINGDOM 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, United Kingdom |
AuthorAffiliation_xml | – name: 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, United Kingdom – name: Imperial College London, UNITED KINGDOM – name: 1 SynthSys Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, United Kingdom – name: 3 Centre for Science at Extreme Condition, University of Edinburgh, Edinburgh, United Kingdom |
Author_xml | – sequence: 1 givenname: Andreas I surname: Andreou fullname: Andreou, Andreas I organization: Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, United Kingdom – sequence: 2 givenname: Naomi orcidid: 0000-0002-9390-3545 surname: Nakayama fullname: Nakayama, Naomi organization: Centre for Science at Extreme Condition, University of Edinburgh, Edinburgh, United Kingdom |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/29293531$$D View this record in MEDLINE/PubMed |
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ContentType | Journal Article |
Copyright | COPYRIGHT 2018 Public Library of Science 2018 Andreou, Nakayama. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. 2018 Andreou, Nakayama 2018 Andreou, Nakayama |
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SubjectTerms | Assembly Automation Biology and Life Sciences Biotechnology Braiding Carotenoids Cassettes Chromobacterium Cloning vectors Construction standards Deoxyribonucleic acid Design Design engineering Design standards DNA DNA - genetics DNA sequencing Domestication E coli Engineering Engineering and Technology Escherichia coli Gene expression Innovations Metabolism Physical Sciences Pigmentation Plant sciences Plasmids Proteins Research and Analysis Methods Standardization Synthetic Biology Technology Transcription |
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Title | Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly |
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