A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/92359 |
Resumo: | Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. |
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A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limitBiochemistry, Genetics and Molecular Biology(all)Agricultural and Biological Sciences(all)Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range.Molecular, Structural and Cellular Microbiology (MOSTMICRO)Instituto de Tecnologia Química e Biológica António Xavier (ITQB)RUNHensel, Zach2020-02-06T23:30:43Z2017-10-012017-10-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10362/92359eng1932-6203PURE: 4073691https://doi.org/10.1371/journal.pone.0187259info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T04:41:10Zoai:run.unl.pt:10362/92359Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:37:31.435976Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
title |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
spellingShingle |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit Hensel, Zach Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) |
title_short |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
title_full |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
title_fullStr |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
title_full_unstemmed |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
title_sort |
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit |
author |
Hensel, Zach |
author_facet |
Hensel, Zach |
author_role |
author |
dc.contributor.none.fl_str_mv |
Molecular, Structural and Cellular Microbiology (MOSTMICRO) Instituto de Tecnologia Química e Biológica António Xavier (ITQB) RUN |
dc.contributor.author.fl_str_mv |
Hensel, Zach |
dc.subject.por.fl_str_mv |
Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) |
topic |
Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) |
description |
Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-10-01 2017-10-01T00:00:00Z 2020-02-06T23:30:43Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/92359 |
url |
http://hdl.handle.net/10362/92359 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1932-6203 PURE: 4073691 https://doi.org/10.1371/journal.pone.0187259 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799137991887683584 |