A "Basic" Approach to the DNA Assembly Bottleneck: A New Standard Designed for Flexibility, Reliability and Automation

The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. We have developed BASIC (Biopart Assembly Standard for Idempotent Cloning), a new physical standard for DNA parts which exploits the principle of orthogonal linker-based DNA assembly. The standard employs a new robust method for assembly, based on type IIs restriction cleavage and ligation of linker oligonucleotides with single stranded overhangs that determine the assembly order. BASIC achieves efficient, parallel assembly with great accuracy: small (4 parts) constructs are routinely assembled with near-100% accuracy, while larger (7 parts) assemblies achieved 90% accuracy. Additionally the linkers can be used as composable parts for RBS tuning or the creation of fusion proteins. This standard format is compatible with a wide range of DNA parts since it only requires a single forbidden restriction site and does not employ any PCR amplifications, and is also conceptually simple at both the design and experimental levels, being based on an idempotent and single-tier organisation where all parts and composite constructs are maintained in the same format. Finally BASIC has been designed with automation in mind, using a workflow that is easily performed on robotic liquid handling platforms.