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.
Highlights
Synthetic biology promises to revolutionize biotechnology by engineering organisms with novel phenotypes not found in nature
We have developed a general modular cloning strategy (MoClo) to allow the systematic assembly of complete eukaryotic transcription units and of multigene constructs from basic premade standardized modules (Fig. 1A)
An assembled transcription unit represents a module again, albeit one of a higher order, which can be directionally assembled into a multigene construct (Fig. 1A)
Summary
Synthetic biology promises to revolutionize biotechnology by engineering organisms with novel phenotypes not found in nature. Since a desired phenotype cannot be predicted directly from gene sequences only, development of strains and optimization of phenotypes will require the ability to generate multiple combinations of various coding sequences as well as many variants of their regulatory sequences. Such optimization does not necessarily need to operate at genome scale, and work currently done for metabolic engineering already belongs to this type of effort. Despite considerable work done in metabolic engineering in the past few years, methods currently used for construct assembly are still limiting, as most of the work is still performed using standard DNA construction techniques that require extensive planning and multiple cloning steps
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