| Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. | |
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MedLine Citation:
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PMID: 22509035 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Bacterial genes associated with a single trait are often grouped in a contiguous unit of the genome known as a gene cluster. It is difficult to genetically manipulate many gene clusters because of complex, redundant, and integrated host regulation. We have developed a systematic approach to completely specify the genetics of a gene cluster by rebuilding it from the bottom up using only synthetic, well-characterized parts. This process removes all native regulation, including that which is undiscovered. First, all noncoding DNA, regulatory proteins, and nonessential genes are removed. The codons of essential genes are changed to create a DNA sequence as divergent as possible from the wild-type (WT) gene. Recoded genes are computationally scanned to eliminate internal regulation. They are organized into operons and placed under the control of synthetic parts (promoters, ribosome binding sites, and terminators) that are functionally separated by spacer parts. Finally, a controller consisting of genetic sensors and circuits regulates the conditions and dynamics of gene expression. We applied this approach to an agriculturally relevant gene cluster from Klebsiella oxytoca encoding the nitrogen fixation pathway for converting atmospheric N(2) to ammonia. The native gene cluster consists of 20 genes in seven operons and is encoded in 23.5 kb of DNA. We constructed a "refactored" gene cluster that shares little DNA sequence identity with WT and for which the function of every genetic part is defined. This work demonstrates the potential for synthetic biology tools to rewrite the genetics encoding complex biological functions to facilitate access, engineering, and transferability. |
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Authors:
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Karsten Temme; Dehua Zhao; Christopher A Voigt |
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Publication Detail:
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Type: Journal Article Date: 2012-04-16 |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 109 ISSN: 1091-6490 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 2012 May |
Date Detail:
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Created Date: 2012-05-02 Completed Date: 2012-07-10 Revised Date: 2013-05-20 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: United States |
Other Details:
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Languages: eng Pagination: 7085-90 Citation Subset: IM |
Affiliation:
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Joint Graduate Group in Bioengineering, University of California, Berkeley/University of California, San Francisco, CA 94158, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence DNA, Bacterial / genetics Gene Expression Regulation, Bacterial Gene Knockout Techniques Genes, Bacterial* Genetic Techniques Klebsiella oxytoca / genetics*, growth & development, metabolism Multigene Family* Nitrogen Fixation / genetics* Nitrogenase / genetics, metabolism Operon Oxidoreductases / genetics, metabolism Plasmids / genetics Promoter Regions, Genetic Ribosomes / metabolism |
| Chemical | |
Reg. No./Substance:
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0/DNA, Bacterial; EC 1.-/Oxidoreductases; EC 1.18.6.1/Nitrogenase; EC 1.18.6.1/nitrogenase reductase |
| Comments/Corrections | |
Comment In:
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Nat Rev Microbiol. 2012 Jun;10(6):378
[PMID:
22580366
]
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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