Multifragment DNA Assembly of Biochemical Pathways via Automated Ligase Cycling Reaction

The microbial production of commodity, fine, and specialty chemicals is a driving force in biotechnology. An essential requirement is to introduce biosynthetic pathways to the target compound(s) into chassis organisms. First suitable enzymes must be selected and characterized, and then genetic pathways must be designed and assembled into suitable expression vectors. The design of these pathways is crucial for balancing the pathway for efficient in vivo activity. This can be achieved through optimization of the pathway regulation by altering transcription and translation rates. The possible permutations of a multigene pathway create a vast design space which is intractable to explore using traditional time-consuming and laborious pathway assembly methods. The advent of multifragment DNA assembly technologies has enabled simultaneous, 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 a workflow that combines automated in silico design of DNA parts followed by pathway assembly using the ligase cycling reaction on robotics platforms, to allow multiplexed assembly of plasmid-borne gene pathways with high efficiency. Details and considerations in designing DNA parts for expression bacterial chassis are discussed followed by laboratory protocols for HTP pathway assembly and screening using robotics platforms. This workflow is employed in the SYNBIOCHEM Synthetic Biology Research Center, providing the capability to assemble over 96 plasmids simultaneously, with over 40% of clones from each assembly harboring the correctly assembled plasmids. This workflow is easy to modify for use in other laboratories and will help to accelerate synthetic biology projects with diverse applications.