Journal
SYNTHETIC BIOLOGY
Volume 5, Issue 1, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/synbio/ysaa001
Keywords
synthetic biology; open source; DNA assembly; openMTA; freedom to use
Funding
- Gordon and Betty Moore Foundation [GBMF5007.01, GBMF4981.01]
- Programa de Apoyo a Centros con Financiamiento Basal [AFB 170004]
- National Institutes of Health [1R01DK11077001A1]
- Instituto Milenio iBio
- Instituto Milenio de Oceanografia of the Iniciativa Cientifica Milenio MINECON [IC120019]
- PUC VRI PhD studentship
- CONICYT [21150778]
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Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a 'common syntax' that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in hostspecific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe 'universal Loop' (uLoop) assembly, a system based on Loop assembly for use in potentially any organismof choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.
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