Publicación:
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales

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dc.contributor.advisorSuárez Barrera, Miguel Orlandospa
dc.contributor.authorAbaunza Villamizar, Sebastián Mauriciospa
dc.date.accessioned2020-01-21T20:43:25Zspa
dc.date.available2020-01-21T20:43:25Zspa
dc.date.issued2018-11-23spa
dc.description91 p.spa
dc.description.abstractBacillus thuringiensis (Bt) is a Gram positive bacterium with parasporal inclusion bodies, also called δ-endotoxins, within these is the Cry protein that is known for its toxic activity against multiple orders of insects and nematodes, the vector-borne diseases A. aegypti, it transmites several important diseases in public health such as Dengue, Zika Chikungunya, among others. Cry11 has been recognized for its high specificity against A. aegypti. However, the emergence of resistance by insects that are the object of study, have resulted in the application of various strategies, for the improvement of proteins including site-directed mutagenesis and DNA shuffling. The use of biocomputational tools has expanded the spectrum in the development of molecules with greater potential, however, these strategies have not been described in the development of enhanced mutants of Cry. This work is based on the use of software HIDDEN 1.0 where libraries of in silico variants were obtained from Cry11Aa. Heu2, Heu3 and Heu4 variants were selected, and validated in vitro, they were synthesized, cloned into PSV2 and were expressed in E.coli DE3BL21 and B. thuringiensis BMB171, their electrophoretic profiles were analyzed in SDS-Page and their toxicity activity was evaluated by thick tests with A. aegypti. Parallel to this a structural analysis with matrix of MatGat, Bioedit and structural comparisons were contrasted with the literature. It was found that the variants presented an upper identity more than 97% with Cry11Aa, the changes mostly belonged to domain II, and their lethality percentages were less than 5%, which suggests that the amino acid changes in the important regions of the three domains are involved in the toxicity activity demonstrated by the proteins.eng
dc.description.abstractBacillus thuringiensis (Bt) es una bacteria Gram positiva con cuerpos de inclusión parasporales, también denominadas δ-endotoxinas, dentro de estas se encuentra la proteína Cry que se conoce por su actividad tóxica frente a múltiples órdenes de insectos y nematodos, dentro de estos A. aegypti principal vector de múltiples enfermedades importantes en la salud pública como Dengue, Zika Chikungunya, entre otros. Cry11 ha sido reconocida por su alta especificidad frente a A. aegypti. Sin embargo, la aparición de resistencias por parte de los insectos que son objeto de estudio, han dado como resultado la aplicación de diversas estrategias, para el mejoramiento de proteínas entre ellas se describe mutagénesis sitio dirigida y DNA shuffling. El uso de herramientas biocomputacionales han ampliado el espectro en el desarrollo de moléculas con mayor potencial, no obstante, estas estrategias no han sido descritas en el desarrollo de mutantes potenciadas de Cry. Este trabajo se basa en el uso del software HIDDEN 1.0 donde a partir de Cry11Aa se obtuvieron librerías de variantes in silico, se seleccionaron las variantes Heu2, Heu3 y Heu4, y se realizó su validación in vitro, para lograrlo se sintetizaron, clonaron en PSV2 y se expresaron en (E.coli) DE3BL21 y BMB171 (B. thuringiensis), se analizó sus perfiles electroforéticos en SDS page y se evaluó su toxicidad mediante ensayos gruesos con A. aegypti, paralelamente se llevó a cabo un análisis estructural con matriz de MatGat, Bioedit y comparaciones estructurales contrastadas con la literatura. Se encontró que las variantes presentaron un porcentaje superior al 97% de identidad con Cry11Aa, los cambios en su mayoría pertenecieron al dominio II, y sus porcentajes de letalidad fueron menor al 5%, lo que sugiere que los cambios aminoacídicos en las regiones importantes de los tres dominios, están involucradas en la toxicidad que presenta la proteína.spa
dc.description.degreelevelPregradospa
dc.description.degreenameMicrobiólogo Industrialspa
dc.description.tableofcontents1. INTRODUCCIÓN ........................................................................... 17 2. PLANTEAMIENTO DEL PROBLEMA ............................................. 21 3.JUSTIFICACIÓN ............................................................................... 24 4. MARCO TEÓRICO ........................................................................... 27 4.1. Generalidades de Bacillus thurigiensis. ...................................... 27 4.2 Proteínas Cry ............................................................................... 28 4.2.1 Dominio I: .............................................................................. 31 4.2.2 Dominio II: ............................................................................. 32 4.2.3 Dominio III: ............................................................................ 33 4.3 Mecanismos de acción de proteínas Cry ..................................... 34 4.3.2 El modelo de vía de señalamiento mediante la formación de canales iónicos.................... 37 4.4 Proteínas Cry 11 .......................................................................... 39 4.5 Modificaciones de toxinas Cry ..................................................... 42 4.5.1 Proteínas Truncadas ............................................................. 42 4.5.2 Modificación en sitios de clivaje ............................................ 43 4.5.3 Modificaciones en los sitios de unión .................................... 44 5. ESTADO DEL ARTE ........................................................................ 45 6. OBJETIVOS ..................................................................................... 49 6.1 OBJETIVO GENERAL ................................................................. 49 6.2 OBJETIVOS ESPECÍFICOS ....................................................... 49 7.7. HIPÓTESISHIPÓTESIS ....................................................................................... 50 8. METODOLOGÍA8. METODOLOGÍA ............................................................................... 51 8.1. Diseño de estudio: ................................................................... 51 8.2. Metodología ............................................................................. 51 8.3 Materiales y MétodosMateriales y Métodos ............................................................... 52 8.3.1 Diseño in silico de genes Cry ................................................ 52 8.3.2 Clonación de genes heurísticos cry en el vector pSV2.......... 52 8.3.3 Transformación en cepas DE3BL21 ...................................... 53 8.4.4 Transformación en cepas BMB171 ....................................... 54 8.4.5 Cultivos de cepas .................................................................. 54 8.4.6 Obtención de cultivos finales de Bt y solubilización de proteínas..................... 55 8.4.7 Cuantificación de proteínas ................................................... 55 8.4.8 Electroforesis de Proteínas ................................................... 56 8.4.9 Estimación peso seco ............................................................ 56 8.4.10 Ensayos de letalidad ............................................................... 56 8.4.12 Análisis estructural .............................................................. 57 8.5 Consideraciones éticas ................................................................ 57 9 RESULTADOS .................................................................................. 58 9.1 Diseño in silico y caracterización de mutantes ............................ 58 9.1.2 Análisis de la secuencia aminoacídica de las variantes. ....... 61 9.2 Subclonaje de variantes Heu 2, Heu3 y Heu4 ............................. 65 9.3 Ensayo Grueso de letalidad ......................................................... 73 10 DISCUSIÓN ..................................................................................... 74 11. CONCLUSIONES ........................................................................... 80 12. RECOMENDACIONES ................................................................... 82 13. BIBLIOGRAFÍA .............................................................................. 83spa
dc.description.versionEj. 1spa
dc.format.mimetypeapplication/pdfspa
dc.identifier.localT 33.18 A118cspa
dc.identifier.urihttps://repositorio.udes.edu.co/handle/001/4341spa
dc.language.isospaspa
dc.publisherBucaramanga : Universidad de Santander, 2018spa
dc.publisher.facultyFacultad de Ciencias Exactas, Naturales y Agropecuariasspa
dc.publisher.programMicrobiología Industrialspa
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dc.rightsDerechos Reservados - Universidad de Santander, 2018spa
dc.rights.accessrightsinfo:eu-repo/semantics/openAccessspa
dc.rights.creativecommonsAtribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)spa
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/spa
dc.subject.proposalBacillus thuringiensisspa
dc.subject.proposalModelos heurísticosspa
dc.subject.proposalCry11Aaspa
dc.subject.proposalA. aegyptispa
dc.subject.proposalHeuristic Modelsspa
dc.titleCaracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionalesspa
dc.typeTrabajo de grado - Pregradospa
dc.type.coarhttp://purl.org/coar/resource_type/c_7a1fspa
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