a modular cloning system for standardized assembly of multigene constructs标准化模块化克隆系统组装的基因结构.pdfVIP

A Modular Cloning System for Standardized Assembly of Multigene Constructs . . Ernst Weber , Carola Engler , Ramona Gruetzner, Stefan Werner, Sylvestre Marillonnet* Icon Genetics GmbH, Halle/Saale, Germany 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. Citation: Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. doi:10.1371/journal.pone.0016765 Editor: Jean Peccoud, Virginia Tech, United States of America Received September 28, 2010; Accepted January 3, 2011; Published February 18, 2011 Copyright: 2011 Weber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unre