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Dual transcriptional-translational cascade permits cellular level tuneable expression control

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription...

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Autores principales: Morra, Rosa, Shankar, Jayendra, Robinson, Christopher J., Halliwell, Samantha, Butler, Lisa, Upton, Mathew, Hay, Sam, Micklefield, Jason, Dixon, Neil
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756846/
https://www.ncbi.nlm.nih.gov/pubmed/26405200
http://dx.doi.org/10.1093/nar/gkv912
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author Morra, Rosa
Shankar, Jayendra
Robinson, Christopher J.
Halliwell, Samantha
Butler, Lisa
Upton, Mathew
Hay, Sam
Micklefield, Jason
Dixon, Neil
author_facet Morra, Rosa
Shankar, Jayendra
Robinson, Christopher J.
Halliwell, Samantha
Butler, Lisa
Upton, Mathew
Hay, Sam
Micklefield, Jason
Dixon, Neil
author_sort Morra, Rosa
collection PubMed
description The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.
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spelling pubmed-47568462016-02-18 Dual transcriptional-translational cascade permits cellular level tuneable expression control Morra, Rosa Shankar, Jayendra Robinson, Christopher J. Halliwell, Samantha Butler, Lisa Upton, Mathew Hay, Sam Micklefield, Jason Dixon, Neil Nucleic Acids Res Methods Online The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. Oxford University Press 2016-02-18 2015-09-23 /pmc/articles/PMC4756846/ /pubmed/26405200 http://dx.doi.org/10.1093/nar/gkv912 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Morra, Rosa
Shankar, Jayendra
Robinson, Christopher J.
Halliwell, Samantha
Butler, Lisa
Upton, Mathew
Hay, Sam
Micklefield, Jason
Dixon, Neil
Dual transcriptional-translational cascade permits cellular level tuneable expression control
title Dual transcriptional-translational cascade permits cellular level tuneable expression control
title_full Dual transcriptional-translational cascade permits cellular level tuneable expression control
title_fullStr Dual transcriptional-translational cascade permits cellular level tuneable expression control
title_full_unstemmed Dual transcriptional-translational cascade permits cellular level tuneable expression control
title_short Dual transcriptional-translational cascade permits cellular level tuneable expression control
title_sort dual transcriptional-translational cascade permits cellular level tuneable expression control
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756846/
https://www.ncbi.nlm.nih.gov/pubmed/26405200
http://dx.doi.org/10.1093/nar/gkv912
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