期刊
ACS SYNTHETIC BIOLOGY
卷 4, 期 9, 页码 1042-1046出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00113
关键词
molecular cloning; DNA assembly; uracil excision cloning; genome engineering
资金
- Novo Nordisk Foundation
- European Union [317058]
- VILLUM Foundation's Young Investigator Programme grant [VKR023128]
- NNF Center for Biosustainability [Microbial Evolution & Synthetic Bio] Funding Source: researchfish
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
- Villum Fonden [00007277] Funding Source: researchfish
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 beta-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.
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