title: A modular cloning system for standardized assembly of multigene constructs.
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.
authors.affiliation: Icon Genetics GmbH, Halle/Saale, Germany.
authors.forename: Ernst
authors.initials: E
authors.lastname: Weber
authors.forename: Carola
authors.initials: C
authors.lastname: Engler
authors.forename: Ramona
authors.initials: R
authors.lastname: Gruetzner
authors.forename: Stefan
authors.initials: S
authors.lastname: Werner
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Marillonnet
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000465:Algorithms; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods* / Q000592:standards*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010941:Plant Tumors / Q000235:genetics / Q000382:microbiology; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012107:Research Design; D014026:Nicotiana / Q000235:genetics / Q000382:microbiology; D019076:Transgenes / Q000502:physiology; D054928:Validation Studies as Topic
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins