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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Cornish, Virginia W. Wingler, Laura M. |
| Description | Author Affiliation: Wingler LM ( Department of Chemistry, Columbia University, 550 West 120th Street, MC 4854, New York, NY 10027, USA. lmw2114@columbia.edu); |
| Abstract | The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop 'Reiterative Recombination,' a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo. |
| ISSN | 00278424 |
| e-ISSN | 10916490 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Issue Number | 37 |
| Volume Number | 108 |
| Language | English |
| Publisher | National Academy of Sciences |
| Publisher Date | 2011-09-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Gene Library Genes, Fungal Genetics Genetic Techniques Multigene Family Recombination, Genetic Saccharomyces Cerevisiae Biosynthetic Pathways Carotenoids Biosynthesis Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S. Multidisciplinary |
| Content Type | Text |
| Resource Type | Article |
| Subject | Multidisciplinary |
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