Multifragment DNA Assembly of Biochemical Pathways via Automated Ligase Cycling Reaction

Methods in Enzymology
Christopher J RobinsonAdrian J Jervis

Abstract

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...Continue Reading

Citations

Jan 22, 2022·ACS Synthetic Biology·Joshua S JamesYizhi Cai

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