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ACS Synth Biol. 2018 Apr 20;7(4):1018-1029. doi: 10.1021/acssynbio.7b00363. Epub 2018 Apr 12.

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

Author information

1
Protein Science & Engineering, Callaghan Innovation, School of Biological Sciences , University of Canterbury , Private Bag 4800, Christchurch 8140 , New Zealand.
2
Department of Chemistry , University of Canterbury , 20 Kirkwood Avenue , Christchurch 8041 , New Zealand.
3
Ferrier Research Institute , Victoria University of Wellington , Kelburn, Wellington 6012 , New Zealand.
4
Institute of Fundamental Sciences , Massey University , Private Bag 11 222, Palmerston North 4442 , New Zealand.

Abstract

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

KEYWORDS:

Golden Gate cloning; Type IIS; indole diterpene; metabolic pathway engineering; modular assembly; synthetic biology

PMID:
29620866
DOI:
10.1021/acssynbio.7b00363
[Indexed for MEDLINE]

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