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Identificación y Caracterización de Parasporinas, Obtenidas de Aislados de Bacillus spp Nativos de Suelos Colombianos

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dc.contributor.advisorSuárez Barrera, Miguel Orlando
dc.contributor.advisorRueda Forero, Nohora Juliana
dc.contributor.authorCastro Pinzón, Yaren Yorlady
dc.date.accessioned2021-08-20T13:48:45Z
dc.date.available2021-08-20T13:48:45Z
dc.date.issued2021-01-28
dc.descriptionDigitalspa
dc.description.abstractA pesar del desarrollo de diversos procedimientos médicos enfocados hacia el tratamiento del cáncer, al día de hoy siguen existiendo falencias en la preservación de tejidos sanos. Por lo anterior, las nuevas estrategias se han centrado en el uso de moléculas biológicas específicas con potencial anticancerígeno. En este sentido, durante los últimos años, han aumentado las investigaciones con Bacillus thuringiensis (Bt), un microorganismo Grampositivo caracterizado por la producción de inclusiones cristalinas que resultan tóxicas hacia varios órdenes de insectos. En los estudios más recientes se ha reportado que Bt también tiene la capacidad de sintetizar proteínas no insecticidas conocidas como Parasporinas (PS), las cuales producen cristales que presentan citotoxicidad contra diferentes líneas celulares de cáncer. El presente proyecto se enfocó en la identificación de genes parasporales en 13 aislamientos de Bacillus spp de la colección del Laboratorio de Biología Molecular y Biotecnología de la Universidad de Santander aisladas de suelos colombianos, por medio de la estandarización de la técnica molecular de reacción en cadena de la polimerasa (PCR). Los análisis en geles de agarosa permitieron determinar la presencia de amplicones de interés para PS1 en los aislados 87 y 88B y para PS2 en el aislado 67. La comparación de la secuencia de aminoácidos del producto de PCR de PS2 con la proteína de referencia PS2Aa1 mostró un porcentaje de identidad del 99%. Así mismo, se evidenció una relación filogenética estrecha entre las secuencias de los amplicones de interés de los aislados 67 y 88B, sugiriendo un alto nivel de conservación entre los genes que codifican PS1 y PS2. Hasta el momento, este es el primer informe de investigaciones con toxinas PS en el territorio colombiano, indicando la existencia de aislados nativos en el país con potencial para la producción de estas proteínas.spa
dc.description.abstractDespite the development of various medical procedures focused on the treatment of cancer, to this day there’re still shortcomings in the preservation of healthy tissues. Therefore, new strategies have focused on the use of specific biological molecules with anticancer potential. In this sense, in recent years, research has increased with Bacillus thuringiensis (Bt), a gram-positive microorganism characterized by the production of crystalline inclusions that are toxic towards various orders of insects. In the most recent studies, it has been reported that Bt also has the ability to synthesize non-insecticidal proteins known as Parasporins (PS), which produce crystals that exhibit cytotoxicity against different cancer cell lines. Recently, 6 families of PS have been identified, based on the homology of their amino acid sequences. This project focused on the identification of parasporal genes in 13 strains of Bacillus spp from the collection of the Molecular Biology and Biotechnology Laboratory of the University of Santander isolated from Colombian soils, through the standardization of polymerase chain reaction technique. (PCR). The analysis in agarose gels made it possible to determine the presence of amplicons of interest for PS1 in isolates 87 and 88B and for PS2 in isolate 67. The analysis of the protein profile using SDS-Page of strain 67 showed the presence of a band 37 kDa, corresponding to the weight of PS2. Similarly, the comparison of the amino acid sequence of this strain with the reference protein PS2Aa1 showed a percentage of identity of 99%. So far, this is the first research report with PS toxins in the Colombian territory, indicating that there’re native isolates in the country with potential for the production of these proteins.eng
dc.description.degreelevelPregradospa
dc.description.degreenameMicrobiólogo Industrialspa
dc.description.edition1 ed.spa
dc.description.tableofcontentsResumen ............................................................................................................................ 12 Abstract ............................................................................................................................. 14 Introducción ...................................................................................................................... 16 1. Objetivos ...................................................................................................................... 20 1.1 Objetivo General .......................................................................................................... 20 1.2 Objetivos Específicos ................................................................................................... 20 2. Pregunta de Investigación ............................................................................................ 21 3. Hipótesis de Investigación............................................................................................ 22 3.1 Hipótesis Nula .............................................................................................................. 22 3.2 Hipótesis Alternativa .................................................................................................... 22 4. Marco Teórico .............................................................................................................. 23 4.1 Bacillus thuringiensis ................................................................................................... 23 4.2 Proteínas Cry ................................................................................................................ 24 4.3 Parasporinas.................................................................................................................. 27 4.3.1 Parasporina 1 (PS1) ...................................................................................................... 29 4.3.2 Parasporina 2 (PS2) ...................................................................................................... 32 4.3.3 Parasporina 3 (PS3) ...................................................................................................... 33 4.3.4 Parasporina 4 (PS4) ...................................................................................................... 35 4.3.5 Parasporina 5 (PS5) ...................................................................................................... 35 4.3.6 Parasporina 6 (PS6) ...................................................................................................... 36 5. Estado del Arte ............................................................................................................. 38 6. Marco Legal ................................................................................................................. 44 7. Materiales y Métodos ................................................................................................... 46 7.1 Diseño del Estudio........................................................................................................ 46 7.2 Metodología.................................................................................................................. 46 7.2.1 Aislamientos Bacterianos y Condiciones de Cultivo ................................................... 46 7.2.2 Caracterización Macroscópica y Microscópica ............................................................ 46 7.2.3 Extracción y Cuantificación de ADN ........................................................................... 47 7.2.4 Cebadores y Condiciones de Amplificación ................................................................ 48 7.2.5 Clonación y Secuenciación Nucleotídica de Amplicones ............................................ 51 7.2.6 Análisis Filogenético .................................................................................................... 52 7.2.7 Extracción Total de Proteínas ....................................................................................... 53 7.2.8 Análisis del Perfil Proteico Mediante SDS-PAGE....................................................... 54 8. Resultados y Discusión ................................................................................................ 55 8.1 Caracterización Macroscópica y Microscópica de Aislamientos de Bacillus spp ....... 55 8.2 Amplificación de Genes ps Mediante PCR .................................................................. 59 8.3 Clonación y Secuenciación Nucleotídica de Amplicones ............................................ 61 8.4 Análisis Filogenético .................................................................................................... 62 8.5 Análisis del Perfil Proteico Mediante SDS-PAGE....................................................... 68 9. Conclusiones ................................................................................................................ 70 Referencias Bibliográficas ................................................................................................ 71 Apéndices .......................................................................................................................... 84spa
dc.format.extent85 pspa
dc.format.mimetypeapplication/pdfspa
dc.identifier.localT 33.21 C188i
dc.identifier.urihttps://repositorio.udes.edu.co/handle/001/5520
dc.language.isospaspa
dc.publisherBucaramanga : Universidad de Santander, 2021spa
dc.publisher.facultyFacultad de Ciencias Exactas, Naturales y Agropecuariasspa
dc.publisher.placeBucaramanga, Colombiaspa
dc.publisher.programMicrobiología Industrialspa
dc.rightsDerechos Reservados - Universidad de Santander, 2021spa
dc.rights.accessrightsinfo:eu-repo/semantics/openAccessspa
dc.rights.creativecommonsAtribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)spa
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/spa
dc.subject.proposalBacillus thuringiensiseng
dc.subject.proposalParasporinasspa
dc.subject.proposalColombiaspa
dc.subject.proposalReacción en Cadena de Polimerasaspa
dc.subject.proposalParasporinseng
dc.subject.proposalPCReng
dc.titleIdentificación y Caracterización de Parasporinas, Obtenidas de Aislados de Bacillus spp Nativos de Suelos Colombianosspa
dc.typeTrabajo de grado - Pregradospa
dc.type.coarhttp://purl.org/coar/resource_type/c_7a1fspa
dc.type.contentTextspa
dc.type.driverinfo:eu-repo/semantics/bachelorThesisspa
dc.type.redcolhttps://purl.org/redcol/resource_type/TPspa
dc.type.versioninfo:eu-repo/semantics/acceptedVersionspa
dcterms.audienceTodas las Audienciasspa
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