Plasmids for Independently Tunable, Low-Noise Expression of Two Genes

ABSTRACT Some microbiology experiments and biotechnology applications can be improved if it is possible to tune the expression of two different genes at the same time with cell-to-cell variation at or below the level of genes constitutively expressed from the chromosome (the “extrinsic noise limit”)...

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Autores principales:	João P. N. Silva, Soraia Vidigal Lopes, Diogo J. Grilo, Zach Hensel
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	expression systems flow cytometry fluorescent-image analysis heterologous gene expression recombinant-protein production regulation of gene expression Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/73882445a67d47499ca3f81000266886
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spelling	oai:doaj.org-article:73882445a67d47499ca3f810002668862021-11-15T15:22:19ZPlasmids for Independently Tunable, Low-Noise Expression of Two Genes10.1128/mSphere.00340-192379-5042https://doaj.org/article/73882445a67d47499ca3f810002668862019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00340-19https://doaj.org/toc/2379-5042ABSTRACT Some microbiology experiments and biotechnology applications can be improved if it is possible to tune the expression of two different genes at the same time with cell-to-cell variation at or below the level of genes constitutively expressed from the chromosome (the “extrinsic noise limit”). This was recently achieved for a single gene by exploiting negative autoregulation by the tetracycline repressor (TetR) and bicistronic gene expression to reduce gene expression noise. We report new plasmids that use the same principles to achieve simultaneous, low-noise expression for two genes in Escherichia coli. The TetR system was moved to a compatible plasmid backbone, and a system based on the lac repressor (LacI) was found to also exhibit gene expression noise below the extrinsic noise limit. We characterized gene expression mean and noise across the range of induction levels for these plasmids, applied the LacI system to tune expression for single-molecule mRNA detection under two different growth conditions, and showed that two plasmids can be cotransformed to independently tune expression of two different genes. IMPORTANCE Microbiologists often express foreign proteins in bacteria in order study them or to use bacteria as a microbial factory. Usually, this requires controlling the number of foreign proteins expressed in each cell, but for many common protein expression systems, it is difficult to “tune” protein expression without large cell-to-cell variation in expression levels (called “noise” in protein expression). This work describes two protein expression systems that can be combined in the same cell, with tunable expression levels and very low protein expression noise. One new system was used to detect single mRNA molecules by fluorescence microscopy, and the two systems were shown to be independent of each other. These protein expression systems may be useful in any experiment or biotechnology application that can be improved with low protein expression noise.João P. N. SilvaSoraia Vidigal LopesDiogo J. GriloZach HenselAmerican Society for Microbiologyarticleexpression systemsflow cytometryfluorescent-image analysisheterologous gene expressionrecombinant-protein productionregulation of gene expressionMicrobiologyQR1-502ENmSphere, Vol 4, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	expression systems flow cytometry fluorescent-image analysis heterologous gene expression recombinant-protein production regulation of gene expression Microbiology QR1-502
spellingShingle	expression systems flow cytometry fluorescent-image analysis heterologous gene expression recombinant-protein production regulation of gene expression Microbiology QR1-502 João P. N. Silva Soraia Vidigal Lopes Diogo J. Grilo Zach Hensel Plasmids for Independently Tunable, Low-Noise Expression of Two Genes
description	ABSTRACT Some microbiology experiments and biotechnology applications can be improved if it is possible to tune the expression of two different genes at the same time with cell-to-cell variation at or below the level of genes constitutively expressed from the chromosome (the “extrinsic noise limit”). This was recently achieved for a single gene by exploiting negative autoregulation by the tetracycline repressor (TetR) and bicistronic gene expression to reduce gene expression noise. We report new plasmids that use the same principles to achieve simultaneous, low-noise expression for two genes in Escherichia coli. The TetR system was moved to a compatible plasmid backbone, and a system based on the lac repressor (LacI) was found to also exhibit gene expression noise below the extrinsic noise limit. We characterized gene expression mean and noise across the range of induction levels for these plasmids, applied the LacI system to tune expression for single-molecule mRNA detection under two different growth conditions, and showed that two plasmids can be cotransformed to independently tune expression of two different genes. IMPORTANCE Microbiologists often express foreign proteins in bacteria in order study them or to use bacteria as a microbial factory. Usually, this requires controlling the number of foreign proteins expressed in each cell, but for many common protein expression systems, it is difficult to “tune” protein expression without large cell-to-cell variation in expression levels (called “noise” in protein expression). This work describes two protein expression systems that can be combined in the same cell, with tunable expression levels and very low protein expression noise. One new system was used to detect single mRNA molecules by fluorescence microscopy, and the two systems were shown to be independent of each other. These protein expression systems may be useful in any experiment or biotechnology application that can be improved with low protein expression noise.
format	article
author	João P. N. Silva Soraia Vidigal Lopes Diogo J. Grilo Zach Hensel
author_facet	João P. N. Silva Soraia Vidigal Lopes Diogo J. Grilo Zach Hensel
author_sort	João P. N. Silva
title	Plasmids for Independently Tunable, Low-Noise Expression of Two Genes
title_short	Plasmids for Independently Tunable, Low-Noise Expression of Two Genes
title_full	Plasmids for Independently Tunable, Low-Noise Expression of Two Genes
title_fullStr	Plasmids for Independently Tunable, Low-Noise Expression of Two Genes
title_full_unstemmed	Plasmids for Independently Tunable, Low-Noise Expression of Two Genes
title_sort	plasmids for independently tunable, low-noise expression of two genes
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/73882445a67d47499ca3f81000266886
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Plasmids for Independently Tunable, Low-Noise Expression of Two Genes

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