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A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria
The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routi...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765039/ https://www.ncbi.nlm.nih.gov/pubmed/29323285 http://dx.doi.org/10.1038/s41598-017-18846-1 |
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author | Horbal, Lilya Siegl, Theresa Luzhetskyy, Andriy |
author_facet | Horbal, Lilya Siegl, Theresa Luzhetskyy, Andriy |
author_sort | Horbal, Lilya |
collection | PubMed |
description | The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device – an in vivo RBS-selector – that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17–100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces. Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria. |
format | Online Article Text |
id | pubmed-5765039 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-57650392018-01-17 A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria Horbal, Lilya Siegl, Theresa Luzhetskyy, Andriy Sci Rep Article The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device – an in vivo RBS-selector – that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17–100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces. Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria. Nature Publishing Group UK 2018-01-11 /pmc/articles/PMC5765039/ /pubmed/29323285 http://dx.doi.org/10.1038/s41598-017-18846-1 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Horbal, Lilya Siegl, Theresa Luzhetskyy, Andriy A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria |
title | A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria |
title_full | A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria |
title_fullStr | A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria |
title_full_unstemmed | A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria |
title_short | A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria |
title_sort | set of synthetic versatile genetic control elements for the efficient expression of genes in actinobacteria |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765039/ https://www.ncbi.nlm.nih.gov/pubmed/29323285 http://dx.doi.org/10.1038/s41598-017-18846-1 |
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