A Modular Cloning System for Standardized Assembly of Multigene Constructs

TLDR

Synthetic biology aims to engineer organisms with novel phenotypes for medicine, agriculture, and industry, yet current DNA assembly methods limit the construction of complex multigene constructs. The authors propose a hierarchical modular cloning system that enables efficient, on‑demand assembly of any eukaryotic multigene construct from validated basic modules. The system employs type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order, facilitating hierarchical modular cloning. Using this approach, the authors assembled a 33‑kb construct containing 11 transcription units from 44 modules in three steps, showing that the MoClo system is automatable and useful for gene stacking and metabolic engineering.

Abstract

The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.