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Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits
E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211737/ https://www.ncbi.nlm.nih.gov/pubmed/25350000 http://dx.doi.org/10.1371/journal.pone.0111451 |
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author | Juhas, Mario Evans, Lewis D. B. Frost, Joe Davenport, Peter W. Yarkoni, Orr Fraser, Gillian M. Ajioka, James W. |
author_facet | Juhas, Mario Evans, Lewis D. B. Frost, Joe Davenport, Peter W. Yarkoni, Orr Fraser, Gillian M. Ajioka, James W. |
author_sort | Juhas, Mario |
collection | PubMed |
description | E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. |
format | Online Article Text |
id | pubmed-4211737 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-42117372014-11-05 Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits Juhas, Mario Evans, Lewis D. B. Frost, Joe Davenport, Peter W. Yarkoni, Orr Fraser, Gillian M. Ajioka, James W. PLoS One Research Article E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. Public Library of Science 2014-10-28 /pmc/articles/PMC4211737/ /pubmed/25350000 http://dx.doi.org/10.1371/journal.pone.0111451 Text en © 2014 Juhas et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Juhas, Mario Evans, Lewis D. B. Frost, Joe Davenport, Peter W. Yarkoni, Orr Fraser, Gillian M. Ajioka, James W. Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits |
title |
Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits |
title_full |
Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits |
title_fullStr |
Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits |
title_full_unstemmed |
Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits |
title_short |
Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits |
title_sort | escherichia coli flagellar genes as target sites for integration and expression of genetic circuits |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211737/ https://www.ncbi.nlm.nih.gov/pubmed/25350000 http://dx.doi.org/10.1371/journal.pone.0111451 |
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