Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication

Abstract

Standardised Type IIS DNA assembly methods are becoming essential for biological engineering and research. Although a 'common syntax' has been proposed to enable higher interoperability between DNA libraries, Golden Gate (GG) -based assembly systems remain specific to target organisms. Furthermore, these GG assembly systems become laborious and unnecessarily complicated beyond the assembly of 4 transcriptional units. Here, we describe "universal Loop" (uLoop) assembly, a simple system based on Loop assembly that enables hierarchical fabrication of large DNA constructs (> 30 kb) for any organism of choice. uLoop comprises two sets of four plasmids that are iteratively used as odd and even levels to compile DNA elements in an exponential manner (4 n-1 ). The elements required for transformation/maintenance in target organisms are also assembled as standardised parts, enabling customisation of host-specific plasmids. Thus, this species-agnostic method decouples efficiency of assembly from the stability of vectors in the target organism. As a proof-of-concept, we show the engineering of multi-gene expression vectors in diatoms, yeast, plants and bacteria. These resources will become available through the OpenMTA for unrestricted sharing and open-access. !

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy

“…Suites of parts specific to microalgae have been developed to be compatible with a common syntax to benefit from existing part registries. Type IIS cloning systems specific to microalgae include the MoClo toolkit for C. reinhardtii (Crozet et al, 2018), CyanoGate for cyanobacteria (Vasudevan et al, 2019), and uLoop for diatoms (Pollak et al, 2019).…”

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

“…As synthetic biology programs increase in scale (1)(2)(3) workflows involving the high-throughput construction of dozens or even hundreds of plasmids are becoming increasingly common (4)(5)(6). DNA sequencing is an integral part of fabrication workflows involving the assembly of synthetic DNA fragments (7)(8)(9)(10).…”

Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

“…Parts are ligated in an ordered manner and restriction sites are removed during assembly, with assemblies of up to 24 parts (7). Different hierarchical standards based on Golden Gate have been published (8)(9)(10)(11)(12)(13)(14)(15)(16)(17), collectively known as modular cloning (or MoClo) standards or toolkits. In MoClo standards, the product of one assembly level can be used as a part in the next level assembly, which is possible due to the use of compatible restriction sites and selection markers in the destination vector ( Figure 1A).…”

“…To allow compatibility of basic parts, multiple toolkits have adopted the PhytoBricks (18) common syntax that dictates that basic parts must be flanked by BsaI with specific fusion sites ( Figure 1B). The PhytoBricks Standard was originally agreed among the plant synthetic biology community but is also used in standards compatible with bacteria (13,14,16). Some MoClo toolkits include special features in their vectors for particular applications.…”

“…PhytoBricks level 0 parts are assembled in any JUMP level 1 vector using BsaI (indicated by the removal of the marker gene); four level 1 products (1A, 1B, 1C, 1D) can be combined in any level 2 vector using BsmBI. As with Mobius(13) and Loop assembly(14,16), JUMP level 1 vectors can be used as assembly destinations for four level 2 products (2A, 2B, 2C, 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.…”