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Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria

Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic t...

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Autores principales: Kelly, Ciarán L., Taylor, George M., Šatkutė, Aistė, Dekker, Linda, Heap, John T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723963/
https://www.ncbi.nlm.nih.gov/pubmed/31426276
http://dx.doi.org/10.3390/microorganisms7080263
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author Kelly, Ciarán L.
Taylor, George M.
Šatkutė, Aistė
Dekker, Linda
Heap, John T.
author_facet Kelly, Ciarán L.
Taylor, George M.
Šatkutė, Aistė
Dekker, Linda
Heap, John T.
author_sort Kelly, Ciarán L.
collection PubMed
description Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and to ‘insulate’ genes and operons from one another. Insulating gene expression can be particularly important when heterologous or synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, Synechocystis sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally–integrated, rhamnose-inducible rhaBAD expression system from hypothesised ‘read-through’ from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.
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spelling pubmed-67239632019-09-10 Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria Kelly, Ciarán L. Taylor, George M. Šatkutė, Aistė Dekker, Linda Heap, John T. Microorganisms Article Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and to ‘insulate’ genes and operons from one another. Insulating gene expression can be particularly important when heterologous or synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, Synechocystis sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally–integrated, rhamnose-inducible rhaBAD expression system from hypothesised ‘read-through’ from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes. MDPI 2019-08-16 /pmc/articles/PMC6723963/ /pubmed/31426276 http://dx.doi.org/10.3390/microorganisms7080263 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kelly, Ciarán L.
Taylor, George M.
Šatkutė, Aistė
Dekker, Linda
Heap, John T.
Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria
title Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria
title_full Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria
title_fullStr Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria
title_full_unstemmed Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria
title_short Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria
title_sort transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723963/
https://www.ncbi.nlm.nih.gov/pubmed/31426276
http://dx.doi.org/10.3390/microorganisms7080263
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