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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.
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 | |
| Publication status | Published - 2015 |
Keywords
- Molecular cloning
- DNA assembly
- Uracil excision cloning
- Genome engineering
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Dive into the research topics of 'Accurate DNA assembly and genome engineering with optimized uracil excision cloning'. Together they form a unique fingerprint.Projects
- 1 Finished
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BacTory: Biorefinery Training Platform: Bacterial Factories for Sustainable Chemical and Drug Production
Molin, S., Nørholm, M., Cavaleiro, M., Machado, H., Wendel, S., van der Helm, E., Bojanovic, K., Vazquez Albacete, D., D'Arrigo, I., Li, S., Yang, X., Redl, S. M. A., Pogrebnyakov, I., Lehning, C. E., Giubergia, S., Calero Valdayo, P. M. & Lohmann, R.
01/10/2012 → 31/01/2017
Project: Research