Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Mafalda Cavaleiro; Se Hyeuk Kim; Susanna Seppala; Morten Thrane Nielsen; Morten H. H. Nørholm

doi:10.1021/acssynbio.5b00113

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Mafalda Cavaleiro
, Se Hyeuk Kim
, Susanna Seppala
, Morten Thrane Nielsen
, Morten H. H. Nørholm

Novo Nordisk Foundation Center for Biosustainability

Research output: Contribution to journal › Journal article › Research › peer-review

Abstract

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.

Original language	English
Journal	A C S Synthetic Biology
Volume	4
Issue number	9
Pages (from-to)	1042-1046
Number of pages	5
ISSN	2161-5063
DOIs	https://doi.org/10.1021/acssynbio.5b00113
Publication status	Published - 2015

Keywords

Molecular cloning
DNA assembly
Uracil excision cloning
Genome engineering

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10.1021/acssynbio.5b00113

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BacTory: Biorefinery Training Platform: Bacterial Factories for Sustainable Chemical and Drug Production
Molin, S. (Project Manager), Nørholm, M. (Project Manager), Cavaleiro, M. (PhD Student), Machado, H. (PhD Student), Wendel, S. (PhD Student), van der Helm, E. (PhD Student), Bojanovic, K. (PhD Student), Vazquez Albacete, D. (Project Participant), D'Arrigo, I. (PhD Student), Li, S. (PhD Student), Yang, X. (PhD Student), Redl, S. M. A. (PhD Student), Pogrebnyakov, I. (PhD Student), Lehning, C. E. (PhD Student), Giubergia, S. (PhD Student), Calero Valdayo, P. M. (PhD Student) & Lohmann, R. (Project Manager)
01/10/2012 → 31/01/2017
Project: Research

Cite this

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title = "Accurate DNA assembly and genome engineering with optimized uracil excision cloning",

abstract = "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.",

keywords = "Molecular cloning, DNA assembly, Uracil excision cloning, Genome engineering",

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AU - Seppala, Susanna

AU - Nielsen, Morten Thrane

AU - Nørholm, Morten H. H.

PY - 2015

Y1 - 2015

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AB - 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.

KW - Molecular cloning

KW - DNA assembly

KW - Uracil excision cloning

KW - Genome engineering

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Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Abstract

Keywords

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Projects

BacTory: Biorefinery Training Platform: Bacterial Factories for Sustainable Chemical and Drug Production

Cite this