Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme

Autores
Andrada Suarez, Eduardo Ezequiel; Sandoval, Evangelina; Fernández Baldo, Martín Alejandro; Cuozzo, Sergio Antonio
Año de publicación
2022
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Streptomyces sp. M7 is an actinobacterium isolated from contaminated sediments with heavy metals and pesticides in Northern Argentina and is able to grow in the presence of hexachlorocyclohexane isomers (γ-HCH, α-HCH, β-HCH) as carbon sources. This property is due to the presence of a high and low metabolic pathway for xenobiotics degradation, where the first enzyme corresponds to LinA, dehydrochlorinase/dehydrohalogenase. This is able to catalyze the breaking of the highly resistant C-Cl bonds of hexachlorocyclohexane, an essential step for its subsequent biodegradation into less toxic metabolic intermediates and/or final products such as carbon dioxide and water. In this work, the linA gene belonging to Streptomyces sp. M7 that codes for dehydrochlorinase (DHC) was synthesized (GenBank: MH703800). The sequence was adapted according to high-frequency-usage codons in E. coli. In the sequence redesign process, two restriction sites, NcoI and XhoI, were added, which are in the bacterial vector pET28a+ used for the expression of 6xHis-tagged N-terminal proteins with a thrombin site. Previously, the synthetic DNA sequence was constructed and cloned in a pTOP Blunt V2 plasmid that was later treated with both restriction enzymes, giving rise to an expected fragment of 475 bp. The released fragment was cloned into the pET28a+ vector and was transformed in competent E. coli BL21 (DE3) host cells with T7 phage promoter. In the same way, transformants were obtained with the expression vector without the cloning fragment and used as control. It should be noted that the plasmid sequence construction and analysis were performed using the Vector NTI software. Subsequently, for the differential expression assay optimization, different conditions were analyzed: 0.4 mM, 0.7 mM, and 1 mM of IPTG, temperatures of 30 and 37 °C and variable times. The most favorable setups were 1 mM of IPTG, 37 °C, and 2 hours of incubation. Subsequently, the cell-free extract was obtained by sonication and analyzed by SDS-PAGE. The stained gels showed a differential band of approximately 39,000 Daltons, confirming the expression of the LinA-6xHis fusion protein. This approach will allow the analysis of the LinA enzyme, as well as its subsequent application in bioremediation technologies.
Fil: Andrada Suarez, Eduardo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Sandoval, Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Fernández Baldo, Martín Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; Argentina
Fil: Cuozzo, Sergio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina
XVII Congreso Argentino de Microbiología General
Los Cocos
Argentina
Sociedad Argentina de Microbiología General
Materia
DECHLORINASE
EXPRESSION
BIOREMEDIATION
LINDANE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/210170

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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzymeAndrada Suarez, Eduardo EzequielSandoval, EvangelinaFernández Baldo, Martín AlejandroCuozzo, Sergio AntonioDECHLORINASEEXPRESSIONBIOREMEDIATIONLINDANEhttps://purl.org/becyt/ford/2.8https://purl.org/becyt/ford/2Streptomyces sp. M7 is an actinobacterium isolated from contaminated sediments with heavy metals and pesticides in Northern Argentina and is able to grow in the presence of hexachlorocyclohexane isomers (γ-HCH, α-HCH, β-HCH) as carbon sources. This property is due to the presence of a high and low metabolic pathway for xenobiotics degradation, where the first enzyme corresponds to LinA, dehydrochlorinase/dehydrohalogenase. This is able to catalyze the breaking of the highly resistant C-Cl bonds of hexachlorocyclohexane, an essential step for its subsequent biodegradation into less toxic metabolic intermediates and/or final products such as carbon dioxide and water. In this work, the linA gene belonging to Streptomyces sp. M7 that codes for dehydrochlorinase (DHC) was synthesized (GenBank: MH703800). The sequence was adapted according to high-frequency-usage codons in E. coli. In the sequence redesign process, two restriction sites, NcoI and XhoI, were added, which are in the bacterial vector pET28a+ used for the expression of 6xHis-tagged N-terminal proteins with a thrombin site. Previously, the synthetic DNA sequence was constructed and cloned in a pTOP Blunt V2 plasmid that was later treated with both restriction enzymes, giving rise to an expected fragment of 475 bp. The released fragment was cloned into the pET28a+ vector and was transformed in competent E. coli BL21 (DE3) host cells with T7 phage promoter. In the same way, transformants were obtained with the expression vector without the cloning fragment and used as control. It should be noted that the plasmid sequence construction and analysis were performed using the Vector NTI software. Subsequently, for the differential expression assay optimization, different conditions were analyzed: 0.4 mM, 0.7 mM, and 1 mM of IPTG, temperatures of 30 and 37 °C and variable times. The most favorable setups were 1 mM of IPTG, 37 °C, and 2 hours of incubation. Subsequently, the cell-free extract was obtained by sonication and analyzed by SDS-PAGE. The stained gels showed a differential band of approximately 39,000 Daltons, confirming the expression of the LinA-6xHis fusion protein. This approach will allow the analysis of the LinA enzyme, as well as its subsequent application in bioremediation technologies.Fil: Andrada Suarez, Eduardo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Sandoval, Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Fernández Baldo, Martín Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; ArgentinaFil: Cuozzo, Sergio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; ArgentinaXVII Congreso Argentino de Microbiología GeneralLos CocosArgentinaSociedad Argentina de Microbiología GeneralSociedad Argentina de Microbiología General2022info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectCongresoBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/210170Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme; XVII Congreso Argentino de Microbiología General; Los Cocos; Argentina; 2022; 73-74CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://samige.org.ar/wp-content/uploads/2022/10/Libro-de-Resumenes-SAMIGE-2022_final.pdfNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:00:33Zoai:ri.conicet.gov.ar:11336/210170instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:00:33.353CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
title Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
spellingShingle Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
Andrada Suarez, Eduardo Ezequiel
DECHLORINASE
EXPRESSION
BIOREMEDIATION
LINDANE
title_short Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
title_full Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
title_fullStr Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
title_full_unstemmed Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
title_sort Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme
dc.creator.none.fl_str_mv Andrada Suarez, Eduardo Ezequiel
Sandoval, Evangelina
Fernández Baldo, Martín Alejandro
Cuozzo, Sergio Antonio
author Andrada Suarez, Eduardo Ezequiel
author_facet Andrada Suarez, Eduardo Ezequiel
Sandoval, Evangelina
Fernández Baldo, Martín Alejandro
Cuozzo, Sergio Antonio
author_role author
author2 Sandoval, Evangelina
Fernández Baldo, Martín Alejandro
Cuozzo, Sergio Antonio
author2_role author
author
author
dc.subject.none.fl_str_mv DECHLORINASE
EXPRESSION
BIOREMEDIATION
LINDANE
topic DECHLORINASE
EXPRESSION
BIOREMEDIATION
LINDANE
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.8
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv Streptomyces sp. M7 is an actinobacterium isolated from contaminated sediments with heavy metals and pesticides in Northern Argentina and is able to grow in the presence of hexachlorocyclohexane isomers (γ-HCH, α-HCH, β-HCH) as carbon sources. This property is due to the presence of a high and low metabolic pathway for xenobiotics degradation, where the first enzyme corresponds to LinA, dehydrochlorinase/dehydrohalogenase. This is able to catalyze the breaking of the highly resistant C-Cl bonds of hexachlorocyclohexane, an essential step for its subsequent biodegradation into less toxic metabolic intermediates and/or final products such as carbon dioxide and water. In this work, the linA gene belonging to Streptomyces sp. M7 that codes for dehydrochlorinase (DHC) was synthesized (GenBank: MH703800). The sequence was adapted according to high-frequency-usage codons in E. coli. In the sequence redesign process, two restriction sites, NcoI and XhoI, were added, which are in the bacterial vector pET28a+ used for the expression of 6xHis-tagged N-terminal proteins with a thrombin site. Previously, the synthetic DNA sequence was constructed and cloned in a pTOP Blunt V2 plasmid that was later treated with both restriction enzymes, giving rise to an expected fragment of 475 bp. The released fragment was cloned into the pET28a+ vector and was transformed in competent E. coli BL21 (DE3) host cells with T7 phage promoter. In the same way, transformants were obtained with the expression vector without the cloning fragment and used as control. It should be noted that the plasmid sequence construction and analysis were performed using the Vector NTI software. Subsequently, for the differential expression assay optimization, different conditions were analyzed: 0.4 mM, 0.7 mM, and 1 mM of IPTG, temperatures of 30 and 37 °C and variable times. The most favorable setups were 1 mM of IPTG, 37 °C, and 2 hours of incubation. Subsequently, the cell-free extract was obtained by sonication and analyzed by SDS-PAGE. The stained gels showed a differential band of approximately 39,000 Daltons, confirming the expression of the LinA-6xHis fusion protein. This approach will allow the analysis of the LinA enzyme, as well as its subsequent application in bioremediation technologies.
Fil: Andrada Suarez, Eduardo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Sandoval, Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Fernández Baldo, Martín Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; Argentina
Fil: Cuozzo, Sergio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina
XVII Congreso Argentino de Microbiología General
Los Cocos
Argentina
Sociedad Argentina de Microbiología General
description Streptomyces sp. M7 is an actinobacterium isolated from contaminated sediments with heavy metals and pesticides in Northern Argentina and is able to grow in the presence of hexachlorocyclohexane isomers (γ-HCH, α-HCH, β-HCH) as carbon sources. This property is due to the presence of a high and low metabolic pathway for xenobiotics degradation, where the first enzyme corresponds to LinA, dehydrochlorinase/dehydrohalogenase. This is able to catalyze the breaking of the highly resistant C-Cl bonds of hexachlorocyclohexane, an essential step for its subsequent biodegradation into less toxic metabolic intermediates and/or final products such as carbon dioxide and water. In this work, the linA gene belonging to Streptomyces sp. M7 that codes for dehydrochlorinase (DHC) was synthesized (GenBank: MH703800). The sequence was adapted according to high-frequency-usage codons in E. coli. In the sequence redesign process, two restriction sites, NcoI and XhoI, were added, which are in the bacterial vector pET28a+ used for the expression of 6xHis-tagged N-terminal proteins with a thrombin site. Previously, the synthetic DNA sequence was constructed and cloned in a pTOP Blunt V2 plasmid that was later treated with both restriction enzymes, giving rise to an expected fragment of 475 bp. The released fragment was cloned into the pET28a+ vector and was transformed in competent E. coli BL21 (DE3) host cells with T7 phage promoter. In the same way, transformants were obtained with the expression vector without the cloning fragment and used as control. It should be noted that the plasmid sequence construction and analysis were performed using the Vector NTI software. Subsequently, for the differential expression assay optimization, different conditions were analyzed: 0.4 mM, 0.7 mM, and 1 mM of IPTG, temperatures of 30 and 37 °C and variable times. The most favorable setups were 1 mM of IPTG, 37 °C, and 2 hours of incubation. Subsequently, the cell-free extract was obtained by sonication and analyzed by SDS-PAGE. The stained gels showed a differential band of approximately 39,000 Daltons, confirming the expression of the LinA-6xHis fusion protein. This approach will allow the analysis of the LinA enzyme, as well as its subsequent application in bioremediation technologies.
publishDate 2022
dc.date.none.fl_str_mv 2022
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Book
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/210170
Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme; XVII Congreso Argentino de Microbiología General; Los Cocos; Argentina; 2022; 73-74
CONICET Digital
CONICET
url http://hdl.handle.net/11336/210170
identifier_str_mv Cloning and expression in E. coli of the linA gene from Streptomyces sp. M7, encoding a dechlorinase enzyme; XVII Congreso Argentino de Microbiología General; Los Cocos; Argentina; 2022; 73-74
CONICET Digital
CONICET
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https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
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dc.publisher.none.fl_str_mv Sociedad Argentina de Microbiología General
publisher.none.fl_str_mv Sociedad Argentina de Microbiología General
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