Universal loop assembly: open, efficient and cross-kingdom DNA fabrication

Abstract

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 host-specific 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 organism of 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.

Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

“…PhytoBrick level 0 parts are assembled in any JUMP level 1 vector (of type 1A, 1B, 1C or 1D) using BsaI (indicated by the removal of the marker gene); four level 1 products (assembled in vectors of type 1A, 1B, 1C and 1D) can be combined in any level 2 vector using BsmBI. As with Mobius ( 15 ) and Loop assembly ( 16 , 18 ), JUMP level 1 vectors can be used as level 3 assembly destinations for four level 2 products (assembled in vectors of type 2A, 2B, 2C and 2D) using BsaI. In addition to the compatibility with other toolkits, the use of constant acceptor sites offers the possibility to introduce sequences from any assembly level into either secondary site in any JUMP vector.…”

“…Basic (level 0) parts in the JUMP toolkit follow the PhytoBrick standard ( 18 ) as shown in Supplementary Figure S2 . Most parts in the toolkit were built from BioBrick parts ( 21 ) or amplified from the genome of E. coli JM109.…”

“…To allow compatibility of basic parts, multiple toolkits have adopted the PhytoBrick ( 20 ) common syntax that dictates that BsaI must flank level 0 parts with specific fusion sites ( Supplementary Figure S2 ). The PhytoBrick Standard was originally agreed among the plant synthetic biology community but is also used in standards compatible with bacteria ( 15 , 16 , 18 ).…”

“…For example, plant toolkits include left and right border sequences flanking the cloning site to allow Agrobacterium -mediated plant transformation ( 10 , 11 , 15 , 16 , 19 ), and some kits include unique flanking nucleotide sequences to allow the combination of multiple assemblies via Gibson assembly ( 16 ). Alternatively, some toolkits extend the possible uses by allowing the addition of features to vectors as additional parts, such as selection markers for target hosts or homology arms for chromosomal integration ( 18 , 19 ). This strategy, however, increases the complexity of the assembly and might require multiple assembly steps.…”

Rapid, robust plasmid verification by de novo assembly of short sequencing reads

“…As synthetic biology programs increase in scale ( 1–3 ) workflows involving the high-throughput construction of dozens or even hundreds of plasmids are becoming increasingly common ( 4 - 6 ). DNA sequencing is an integral part of fabrication workflows involving the assembly of synthetic DNA fragments ( 7–10 ).…”