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Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli

In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been prove...

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Detalles Bibliográficos
Autores principales: Nakashima, Nobutaka, Tamura, Tomohiro
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731896/
https://www.ncbi.nlm.nih.gov/pubmed/19515932
http://dx.doi.org/10.1093/nar/gkp498
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author Nakashima, Nobutaka
Tamura, Tomohiro
author_facet Nakashima, Nobutaka
Tamura, Tomohiro
author_sort Nakashima, Nobutaka
collection PubMed
description In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications.
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spelling pubmed-27318962009-09-10 Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli Nakashima, Nobutaka Tamura, Tomohiro Nucleic Acids Res Methods Online In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications. Oxford University Press 2009-08 2009-06-10 /pmc/articles/PMC2731896/ /pubmed/19515932 http://dx.doi.org/10.1093/nar/gkp498 Text en © 2009 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Nakashima, Nobutaka
Tamura, Tomohiro
Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli
title Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli
title_full Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli
title_fullStr Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli
title_full_unstemmed Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli
title_short Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli
title_sort conditional gene silencing of multiple genes with antisense rnas and generation of a mutator strain of escherichia coli
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731896/
https://www.ncbi.nlm.nih.gov/pubmed/19515932
http://dx.doi.org/10.1093/nar/gkp498
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