Noise Minimization in Cell-Free Gene Expression

Mart W. Bartelds; Óscar García-Blay; Pieter G.A. Verhagen; Elise J. Wubbolts; Bob van Sluijs; Hans A. Heus; Tom F.A. de Greef; Wilhelm T.S. Huck; Maike M.K. Hansen

doi:10.1021/acssynbio.3c00174

Noise Minimization in Cell-Free Gene Expression

Mart W. Bartelds
, Óscar García-Blay
, Pieter G.A. Verhagen
, Elise J. Wubbolts
, Bob van Sluijs
, Hans A. Heus
, Tom F.A. de Greef
, Wilhelm T.S. Huck
, Maike M.K. Hansen (Corresponding author)

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise >2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.

Original language	English
Pages (from-to)	2217-2225
Number of pages	9
Journal	ACS Synthetic Biology
Volume	12
Issue number	8
DOIs	https://doi.org/10.1021/acssynbio.3c00174
Publication status	Published - 18 Aug 2023

Bibliographical note

Funding Information:
We thank Aigars Piruska for making the silicon wafers and providing advice related to the microscope setup, Pascal Pieters for providing the E. coli BL21(Star) strain, and Maaruthy Yelleswarapu for providing the T7p14-mCherry template. T.F.A.d.G. acknowledges funding from the European Research Council (ERC Project 101000199 AMIGA) and the Alliance Center of Living Technologies (CLT). W.T.S.H. acknowledges support through the “BaSyC–Building a Synthetic Cell” Gravitation Grant (024.003.019) of the Ministry of Education, Culture and Science. M.M.K.H. acknowledges support from Radboud University, the Christine Mohrmann Foundation, and The Netherlands Organization for Scientific Research (NWO ENW-XS Awards OCENW.XS3.055 and OCENW.XS22.2.149).

Funding

We thank Aigars Piruska for making the silicon wafers and providing advice related to the microscope setup, Pascal Pieters for providing the E. coli BL21(Star) strain, and Maaruthy Yelleswarapu for providing the T7p14-mCherry template. T.F.A.d.G. acknowledges funding from the European Research Council (ERC Project 101000199 AMIGA) and the Alliance Center of Living Technologies (CLT). W.T.S.H. acknowledges support through the \u201CBaSyC-Building a Synthetic Cell\u201D Gravitation Grant (024.003.019) of the Ministry of Education, Culture and Science. M.M.K.H. acknowledges support from Radboud University, the Christine Mohrmann Foundation, and The Netherlands Organization for Scientific Research (NWO ENW-XS Awards OCENW.XS3.055 and OCENW.XS22.2.149). We thank Aigars Piruska for making the silicon wafers and providing advice related to the microscope setup, Pascal Pieters for providing the E. coli BL21(Star) strain, and Maaruthy Yelleswarapu for providing the T7p14-mCherry template. T.F.A.d.G. acknowledges funding from the European Research Council (ERC Project 101000199 AMIGA) and the Alliance Center of Living Technologies (CLT). W.T.S.H. acknowledges support through the \u201CBaSyC\u2013Building a Synthetic Cell\u201D Gravitation Grant (024.003.019) of the Ministry of Education, Culture and Science. M.M.K.H. acknowledges support from Radboud University, the Christine Mohrmann Foundation, and The Netherlands Organization for Scientific Research (NWO ENW-XS Awards OCENW.XS3.055 and OCENW.XS22.2.149).

Keywords

cell-free gene expression
gene expression noise
MazF
microfluidics
mRNA degradation
transcription and translation

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title = "Noise Minimization in Cell-Free Gene Expression",

abstract = "Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise >2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.",

keywords = "cell-free gene expression, gene expression noise, MazF, microfluidics, mRNA degradation, transcription and translation",

author = "Bartelds, \{Mart W.\} and {\'O}scar Garc{\'i}a-Blay and Verhagen, \{Pieter G.A.\} and Wubbolts, \{Elise J.\} and \{van Sluijs\}, Bob and Heus, \{Hans A.\} and \{de Greef\}, \{Tom F.A.\} and Huck, \{Wilhelm T.S.\} and Hansen, \{Maike M.K.\}",

note = "Funding Information: We thank Aigars Piruska for making the silicon wafers and providing advice related to the microscope setup, Pascal Pieters for providing the E. coli BL21(Star) strain, and Maaruthy Yelleswarapu for providing the T7p14-mCherry template. T.F.A.d.G. acknowledges funding from the European Research Council (ERC Project 101000199 AMIGA) and the Alliance Center of Living Technologies (CLT). W.T.S.H. acknowledges support through the {\textquotedblleft}BaSyC{\textendash}Building a Synthetic Cell{\textquotedblright} Gravitation Grant (024.003.019) of the Ministry of Education, Culture and Science. M.M.K.H. acknowledges support from Radboud University, the Christine Mohrmann Foundation, and The Netherlands Organization for Scientific Research (NWO ENW-XS Awards OCENW.XS3.055 and OCENW.XS22.2.149). ",

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month = aug,

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doi = "10.1021/acssynbio.3c00174",

language = "English",

volume = "12",

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journal = "ACS Synthetic Biology",

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T1 - Noise Minimization in Cell-Free Gene Expression

AU - Bartelds, Mart W.

AU - García-Blay, Óscar

AU - Verhagen, Pieter G.A.

AU - Wubbolts, Elise J.

AU - van Sluijs, Bob

AU - Heus, Hans A.

AU - de Greef, Tom F.A.

AU - Huck, Wilhelm T.S.

AU - Hansen, Maike M.K.

N1 - Funding Information: We thank Aigars Piruska for making the silicon wafers and providing advice related to the microscope setup, Pascal Pieters for providing the E. coli BL21(Star) strain, and Maaruthy Yelleswarapu for providing the T7p14-mCherry template. T.F.A.d.G. acknowledges funding from the European Research Council (ERC Project 101000199 AMIGA) and the Alliance Center of Living Technologies (CLT). W.T.S.H. acknowledges support through the “BaSyC–Building a Synthetic Cell” Gravitation Grant (024.003.019) of the Ministry of Education, Culture and Science. M.M.K.H. acknowledges support from Radboud University, the Christine Mohrmann Foundation, and The Netherlands Organization for Scientific Research (NWO ENW-XS Awards OCENW.XS3.055 and OCENW.XS22.2.149).

PY - 2023/8/18

Y1 - 2023/8/18

N2 - Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise >2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.

AB - Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise >2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.

KW - cell-free gene expression

KW - gene expression noise

KW - MazF

KW - microfluidics

KW - mRNA degradation

KW - transcription and translation

UR - https://www.scopus.com/pages/publications/85166750422

U2 - 10.1021/acssynbio.3c00174

DO - 10.1021/acssynbio.3c00174

M3 - Article

C2 - 37478000

AN - SCOPUS:85166750422

SN - 2161-5063

VL - 12

SP - 2217

EP - 2225

JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

IS - 8

ER -

Noise Minimization in Cell-Free Gene Expression

Abstract

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Keywords

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