Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning

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• 作者：Ana Mafalda Cavaleiro ; Se Hyeuk Kim ; Susanna Sepp盲l盲 ; Morten T. Nielsen ; Morten H. H. N酶rholm
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：September 18, 2015
• 年：2015
• 卷：4
• 期：9
• 页码：1042-1046
• 全文大小：378K
• ISSN：2161-5063

文摘

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that produces 尾-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories.