Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus

La ADN polimerasa de Geobacillus stearothermophilus (ADN Pol I Bst), es un miembro de la familia A de las polimerasas, que posee características desplazamiento de hebra que favorecen su aplicación en métodos de amplificación isotérmica. En el presente trabajo se describe la expresión y caracterizaci...

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Autores:: Estupiñan Molina, Cristian David

Tipo de recurso:

Fecha de publicación:: 2024

Institución:: Universidad Nacional de Colombia

Repositorio:: Universidad Nacional de Colombia

Idioma:: spa

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network_acronym_str	UNACIONAL2
network_name_str	Universidad Nacional de Colombia
repository_id_str
dc.title.spa.fl_str_mv	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
dc.title.translated.eng.fl_str_mv	Expression and functional characterization of a DNA polymerase I from Geobacillus stearothermophilus
title	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
spellingShingle	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus 540 - Química y ciencias afines 610 - Medicina y salud::616 - Enfermedades 570 - Biología::572 - Bioquímica ADN Polimerasa I Polimerasa Taq Virus de la Mieloblastosis Aviar Coronavirus Prueba de COVID-19 DNA Polymerase I Taq Polymerase Avian Myeloblastosis Virus COVID-19 Testing Geobacillus stearothermophilus Polimerasa ADN Pol I Bst Pol Bst LAMP SARS-CoV-2 Polyerase Pol I Bst DNA
title_short	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
title_full	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
title_fullStr	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
title_full_unstemmed	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
title_sort	Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus
dc.creator.fl_str_mv	Estupiñan Molina, Cristian David
dc.contributor.advisor.none.fl_str_mv	de Brito Brandão, Pedro Filipe Calderón Manrique, Dayana
dc.contributor.author.none.fl_str_mv	Estupiñan Molina, Cristian David
dc.contributor.researchgroup.spa.fl_str_mv	Biotecnología Molecular (CorpoGen) Grupo de Estudios para la Remediación y Mitigación de Impactos Negativos al Ambiente (GERMINA)
dc.contributor.cvlac.spa.fl_str_mv	https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000051741
dc.contributor.researchgate.spa.fl_str_mv	https://www.researchgate.net/profile/Cristian-Estupinan
dc.subject.ddc.spa.fl_str_mv	540 - Química y ciencias afines 610 - Medicina y salud::616 - Enfermedades 570 - Biología::572 - Bioquímica
topic	540 - Química y ciencias afines 610 - Medicina y salud::616 - Enfermedades 570 - Biología::572 - Bioquímica ADN Polimerasa I Polimerasa Taq Virus de la Mieloblastosis Aviar Coronavirus Prueba de COVID-19 DNA Polymerase I Taq Polymerase Avian Myeloblastosis Virus COVID-19 Testing Geobacillus stearothermophilus Polimerasa ADN Pol I Bst Pol Bst LAMP SARS-CoV-2 Polyerase Pol I Bst DNA
dc.subject.decs.spa.fl_str_mv	ADN Polimerasa I Polimerasa Taq Virus de la Mieloblastosis Aviar Coronavirus Prueba de COVID-19
dc.subject.decs.eng.fl_str_mv	DNA Polymerase I Taq Polymerase Avian Myeloblastosis Virus COVID-19 Testing
dc.subject.proposal.spa.fl_str_mv	Geobacillus stearothermophilus Polimerasa ADN Pol I Bst Pol Bst LAMP SARS-CoV-2
dc.subject.proposal.eng.fl_str_mv	Polyerase Pol I Bst DNA
description	La ADN polimerasa de Geobacillus stearothermophilus (ADN Pol I Bst), es un miembro de la familia A de las polimerasas, que posee características desplazamiento de hebra que favorecen su aplicación en métodos de amplificación isotérmica. En el presente trabajo se describe la expresión y caracterización funcional de una ADN Pol I Bst, iniciando por la secuenciación del plásmido mediante tecnología Oxford Nanopore, para confirmar la secuencia codificante de la proteína, seguido de un análisis in sillico, con el propósito de determinar la estructura 3D y sitios activos de la proteína. Posteriormente, se realizó la expresión, extracción, purificación, determinación de la actividad catalítica y análisis de funcionalidad mediante Loop Mediated Isothermal Amplification (LAMP) a 65 °C por 60 min, utilizando cómo sustrato ARN de SARS-CoV-2. Los resultados reflejan una secuencia codificante de 576 aminoácidos que pertenece al fragmento grande de ADN Pol I Bst, el cual tiene peso molecular de 61,8 kDa. Se obtuvo una concentración de 2mg/ml de proteína total, que posee una actividad enzimática de 606.4 U y una actividad especifica de 3.0×105 U/mg. Finalmente, se demuestra que la proteína es funcional al amplificar una secuencia perteneciente al Orf1a de ARN de SARS-CoV-2. Aquí se presenta una proteína funcional, con libertad de operación para su distribución y que es aplicable a sistemas de amplificación isotérmica para el diagnóstico de enfermedades de importancia clínica (Texto tomado de la fuente).
publishDate	2024
dc.date.accessioned.none.fl_str_mv	2024-07-16T16:33:17Z
dc.date.available.none.fl_str_mv	2024-07-16T16:33:17Z
dc.date.issued.none.fl_str_mv	2024
dc.type.spa.fl_str_mv	Trabajo de grado - Maestría
dc.type.driver.spa.fl_str_mv	info:eu-repo/semantics/masterThesis
dc.type.version.spa.fl_str_mv	info:eu-repo/semantics/acceptedVersion
dc.type.content.spa.fl_str_mv	Text
dc.type.redcol.spa.fl_str_mv	http://purl.org/redcol/resource_type/TM
status_str	acceptedVersion
dc.identifier.uri.none.fl_str_mv	https://repositorio.unal.edu.co/handle/unal/86461
dc.identifier.instname.spa.fl_str_mv	Universidad Nacional de Colombia
dc.identifier.reponame.spa.fl_str_mv	Repositorio Institucional Universidad Nacional de Colombia
dc.identifier.repourl.spa.fl_str_mv	https://repositorio.unal.edu.co/
url	https://repositorio.unal.edu.co/handle/unal/86461 https://repositorio.unal.edu.co/
identifier_str_mv	Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia
dc.language.iso.spa.fl_str_mv	spa
language	spa
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spelling	Reconocimiento 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2de Brito Brandão, Pedro Filipe9ec73d77d555a4dff5e0457f24f26aefCalderón Manrique, Dayana98bcf96f58fa987f66eec1c797627942Estupiñan Molina, Cristian David8e668d50981c5137d386befa38458f06Biotecnología Molecular (CorpoGen)Grupo de Estudios para la Remediación y Mitigación de Impactos Negativos al Ambiente (GERMINA)https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000051741https://www.researchgate.net/profile/Cristian-Estupinan2024-07-16T16:33:17Z2024-07-16T16:33:17Z2024https://repositorio.unal.edu.co/handle/unal/86461Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/La ADN polimerasa de Geobacillus stearothermophilus (ADN Pol I Bst), es un miembro de la familia A de las polimerasas, que posee características desplazamiento de hebra que favorecen su aplicación en métodos de amplificación isotérmica. En el presente trabajo se describe la expresión y caracterización funcional de una ADN Pol I Bst, iniciando por la secuenciación del plásmido mediante tecnología Oxford Nanopore, para confirmar la secuencia codificante de la proteína, seguido de un análisis in sillico, con el propósito de determinar la estructura 3D y sitios activos de la proteína. Posteriormente, se realizó la expresión, extracción, purificación, determinación de la actividad catalítica y análisis de funcionalidad mediante Loop Mediated Isothermal Amplification (LAMP) a 65 °C por 60 min, utilizando cómo sustrato ARN de SARS-CoV-2. Los resultados reflejan una secuencia codificante de 576 aminoácidos que pertenece al fragmento grande de ADN Pol I Bst, el cual tiene peso molecular de 61,8 kDa. Se obtuvo una concentración de 2mg/ml de proteína total, que posee una actividad enzimática de 606.4 U y una actividad especifica de 3.0×105 U/mg. Finalmente, se demuestra que la proteína es funcional al amplificar una secuencia perteneciente al Orf1a de ARN de SARS-CoV-2. Aquí se presenta una proteína funcional, con libertad de operación para su distribución y que es aplicable a sistemas de amplificación isotérmica para el diagnóstico de enfermedades de importancia clínica (Texto tomado de la fuente).The DNA polymerase from Geobacillus stearothermophilus (Bst DNA Pol I) is a member of the A family of polymerases, exhibiting strand displacement characteristics that favor its application in isothermal amplification methods. This study describes the expression and functional characterization of a Bst DNA Pol I, starting with plasmid sequencing using Oxford Nanopore technology to confirm the protein's coding sequence. This is followed by in silico analysis to determine the protein's 3D structure and active sites. Subsequently, expression, extraction, purification, determination of catalytic activity, and functionality analysis were performed using Loop-Mediated Isothermal Amplification (LAMP) at 65 °C for 60 min, with SARS-CoV-2 RNA as the substrate. The results reveal a coding sequence of 576 amino acids belonging to the large fragment of Bst DNA Pol I, with a molecular weight of 61.8 kDa. A protein concentration of 2 mg/ml was obtained, exhibiting enzymatic activity of 606.4 U and a specific activity of 3.0×105 U/mg. Finally, it is demonstrated that the protein is functional in amplifying a sequence belonging to the Orf1a of SARS-CoV-2 RNA. While optimization studies are important to enhance the protein production process, this study presents a functional protein with operational freedom for distribution and applicability in isothermal amplification systems for the diagnosis of clinically significant diseases.Ministerio de Ciencia Tecnología e InnovaciónMaestríaMagíster en Ciencias - BioquímicaEn este proyecto se realizó la expresión y caracterización funcional de una ADN Pol I Bst, a partir de una plásmido previamente clonado. El enfoque metodológico abarca diferentes etapas, iniciando por la secuenciación del plásmido mediante tecnología Oxford Nanopore (Kwok et al., 2014) seguido de análisis secuencias y el procedimiento para el análisis in sillico, con el propósito de determinar la estructura 3D y sitios activos de la proteína. Posteriormente se detalla la metodología de los objetivos planteados que incluyen la expresión, extracción, purificación, determinación de actividad catalítica y, finalmente, el análisis de funcionalidad utilizando como sustrato ARN de SARS-CoV-2.Desarrollo de Productos Biotecnológicosxiv, 60 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - BioquímicaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá540 - Química y ciencias afines610 - Medicina y salud::616 - Enfermedades570 - Biología::572 - BioquímicaADN Polimerasa IPolimerasa TaqVirus de la Mieloblastosis AviarCoronavirusPrueba de COVID-19DNA Polymerase ITaq PolymeraseAvian Myeloblastosis VirusCOVID-19 TestingGeobacillus stearothermophilusPolimerasaADN Pol I BstPol BstLAMPSARS-CoV-2PolyerasePol I Bst DNAExpresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilusExpression and functional characterization of a DNA polymerase I from Geobacillus stearothermophilusTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMAgustriana, E., Nuryana, I., Laksmi, F. 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The New England Journal of Medicine, 382(8), 727. https://doi.org/10.1056/NEJMOA2001017BibliotecariosEstudiantesGrupos comunitariosInvestigadoresMaestrosPadres y familiasPersonal de apoyo escolarPúblico generalLICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/86461/1/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD51ORIGINAL1069262590.2024.pdf1069262590.2024.pdfTesis de Maestría en Ciencias-Bioquímicaapplication/pdf1679458https://repositorio.unal.edu.co/bitstream/unal/86461/2/1069262590.2024.pdf7f465f2339a4cc21a202b6f55524335bMD52THUMBNAIL1069262590.2024.pdf.jpg1069262590.2024.pdf.jpgGenerated Thumbnailimage/jpeg4835https://repositorio.unal.edu.co/bitstream/unal/86461/3/1069262590.2024.pdf.jpg4c7516d57f46b3c930ad0f3dad28af25MD53unal/86461oai:repositorio.unal.edu.co:unal/864612024-07-16 23:05:05.645Repositorio Institucional Universidad Nacional de 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Expresión y caracterización funcional de una ADN polimerasa I de Geobacillus stearothermophilus

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