Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning.
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 β-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.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, DK-2970 Hørsholm, Denmark.
Ana Mafalda
AM
Cavaleiro
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, DK-2970 Hørsholm, Denmark.
Se Hyeuk
SH
Kim
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, DK-2970 Hørsholm, Denmark.
Susanna
S
Seppälä
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, DK-2970 Hørsholm, Denmark.
Morten T
MT
Nielsen
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, DK-2970 Hørsholm, Denmark.
Morten H H
MH
Nørholm
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000235:genetics*; D005818:Genetic Engineering; D005810:Multigene Family; D020636:Pantoea / Q000235:genetics*; D014498:Uracil / Q000737:chemistry*; D019207:beta Carotene / Q000096:biosynthesis
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D019207:beta Carotene; D014498:Uracil
DNA assembly; genome engineering; molecular cloning; uracil excision cloning