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Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris

BACKGROUND: Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production. RESULTS: Here we present five no...

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Autores principales: Delic, Marizela, Mattanovich, Diethard, Gasser, Brigitte
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599224/
https://www.ncbi.nlm.nih.gov/pubmed/23347582
http://dx.doi.org/10.1186/1475-2859-12-6
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author Delic, Marizela
Mattanovich, Diethard
Gasser, Brigitte
author_facet Delic, Marizela
Mattanovich, Diethard
Gasser, Brigitte
author_sort Delic, Marizela
collection PubMed
description BACKGROUND: Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production. RESULTS: Here we present five novel repressible Pichia pastoris promoters of different strength: P(SER1), P(MET3), P(THR1), P(PIS1) and P(THI11). eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated. CONCLUSION: Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength.
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spelling pubmed-35992242013-03-17 Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris Delic, Marizela Mattanovich, Diethard Gasser, Brigitte Microb Cell Fact Technical Notes BACKGROUND: Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production. RESULTS: Here we present five novel repressible Pichia pastoris promoters of different strength: P(SER1), P(MET3), P(THR1), P(PIS1) and P(THI11). eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated. CONCLUSION: Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength. BioMed Central 2013-01-24 /pmc/articles/PMC3599224/ /pubmed/23347582 http://dx.doi.org/10.1186/1475-2859-12-6 Text en Copyright ©2013 Delic et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Technical Notes
Delic, Marizela
Mattanovich, Diethard
Gasser, Brigitte
Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
title Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
title_full Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
title_fullStr Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
title_full_unstemmed Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
title_short Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
title_sort repressible promoters – a novel tool to generate conditional mutants in pichia pastoris
topic Technical Notes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599224/
https://www.ncbi.nlm.nih.gov/pubmed/23347582
http://dx.doi.org/10.1186/1475-2859-12-6
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