Multi-fragment DNA assembly of biochemical pathways via automated Ligase Cycling Reaction

The microbial production of commodity, fine and specialty chemicals and biofuels is a driving force in biotechnology. To do this, biochemical pathways to the target compound(s) must be deduced, suitable enzymes selected and then the genetic pathways must be designed and built for in vivo activity. The genetic design is crucial for balancing the pathway in vivo through regulation of transcription and translation but the possible permutations quickly generates a vast design space. Traditionally pathway assembly has been time-consuming and laborious but the advent of multi-fragment DNA assembly technologies has facilitated the possibility of multiplexed pathway construction allowing an increased capability to sample the design space. Furthermore, the implementation of laboratory automation allows error-reduced, high-throughput (HTP) construction of pathways. In this chapter we present an automated workflow that combines in silico design of DNA parts followed by pathway assembly using the Ligase Cycling Reaction (LCR) on robotics platforms, to allow multiplexed assembly of plasmid-borne gene pathways with high efficiency. The workflow begins with the design of DNA part sequences considering biological issues and ensuring compatibility with DNA synthesis and LCR assembly. Subsequently a laboratory protocol for HTP pathway assembly and screening is detailed allowing the production of over 96 plasmids simultaneously with a success rate of over 40 %. This workflow is easy to modify for other laboratories and will help to accelerate synthetic biology for diverse application.