Análisis estructural y determinación de la actividad tóxica de las mutantes 8cry11l553f, 8cry11l556w, y 8cry11l553f-l556w obtenidas por mutagénesis sitio dirigida en larvas de primer estadio de aedes aegypti.

dc.contributorSuárez Barrera, Miguel Orlando
dc.contributorRueda Forero, Nohora Juliana
dc.creatorHerrera Pineda, Diego Fernando
dc.date.accessioned2020-01-21T21:10:20Z
dc.date.available2020-01-21T21:10:20Z
dc.date.created2020-01-21T21:10:20Z
dc.date.issued2018-11-23
dc.identifierT 33.18 H277a
dc.identifierhttps://repositorio.udes.edu.co/handle/001/4342
dc.description.abstractBacillus thuringiensis is a Gram-positive bacterium, δ-endotoxins producer that are toxic to different orders of insects and nematodes. Cry11 is a specific toxin against the vector A. aegypti, which is responsible for the transmission of dengue, zika and chikungunya; however, its mode of action and structure-function characteristics have not yet been fully elucidated. The research group of Molecular Biology and Biotechnology of the UDES, has a library obtained by shuffling the DNA of cry11 genes, highlighting the variant 8Cry11, which is 6 times more toxic than Cry11Aa and 3.8 more than Cry11Bb. Molecular Docking studies showed that positions 553 and 556 of this protein are relevant in the interaction with the cadherin receptor, to corroborate this information, site-directed mutagenesis was performed to reverse the aforementioned mutations, obtaining the variants 8Cry11L553F, 8Cry11L556W, and 8Cry11L553F-L556W. In this work, the toxic activity of mutants 8Cry11L553F, 8Cry11L556W, and 8Cry11L553F-L556W was determined, as well as an approximation of protein analysis both in silico and in vitro through SDS-PAGE. To achieve this, the ideal conditions for the production of δ-endotoxin (Cry11Aa) were standardized, finding a relationship between glucose concentrations (15g / L) and sources of organic and inorganic nitrogen in a ratio of 3: 7; the production of protoxin (~ 100 kDa) and toxin (32 and 34 kDa) was corroborated by SDS page. To determine the mean lethal concentration in comparison with the mutant 8Cry11 and the parental Cry11Aa, the toxicity of the mutants was evaluated, against first stage larvae of A. aegypti. The results showed loss of toxicity for the variants under study, which indicated that substituted amino acids in domain III were strongly possible involved in the loss of toxicity due to structural features.
dc.description.abstractBacillus thuringiensis es un bacilo Gram-positivo productor de δ-endotoxinas que son tóxicas para diferentes órdenes de insectos y nematodos. Cry11 es una toxina específica contra el vector A. aegypti, el cual es el responsable de la trasmisión del dengue, zika y chikungunya; sin embargo, su modo de acción y características estructura-función aún no se han elucidado completamente. El grupo de investigación de Biología Molecular y Biotecnología de la UDES, cuenta con una librería obtenida por barajado de ADN de genes cry11, resaltando la variante 8Cry11, la cual es 6 veces más toxica que Cry11Aa y 3,8 más que Cry11Bb. Estudios de Docking molecular demostraron que las posiciones 553 y 556 de esta proteína son relevantes en la interacción con el receptor cadherina, para corroborar esta información se realizó mutagénesis sitio dirigida para revertir las mutaciones mencionadas, obteniendo las variantes 8Cry11L553F, 8Cry11L556W, y 8Cry11L553F-L556W. En este trabajo se determinó la actividad tóxica de las mutantes 8Cry11L553F, 8Cry11L556W, y 8Cry11L553F-L556W así como una aproximación de análisis de proteínas tanto in silico como in vitro a través de SDS-PAGE. Para lograrlo se estandarizó las condiciones ideales para la producción de la δ-endotoxina (Cry11Aa), encontrándose una relación entre las concentraciones de glucosa (15g/L) y las fuentes de nitrógeno orgánico e inorgánico en una proporción de 3:7; se corroboró mediante SDS page la producción de protoxina (~100 kDa) y toxina (32 y 34 kDa). Se evaluó la toxicidad de las mutantes frente a larvas en primer estadío de A. aegypti para determinar la concentración letal media en comparación con la mutante 8Cry11 y la parental Cry11Aa, los resultados mostraron pérdida de toxicidad para las variantes en estudio, lo cual indicó que aminoácidos sustituidos en el dominio III fueron los posibles involucrados en la perdida de la toxicidad debido a la características estructurales.
dc.languagespa
dc.publisherBucaramanga : Universidad de Santander, 2018
dc.publisherFacultad de Ciencias Exactas, Naturales y Agropecuarias
dc.publisherMicrobiología Industrial
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dc.rightsinfo:eu-repo/semantics/openAccess
dc.rightsAtribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)
dc.rightshttps://creativecommons.org/licenses/by-nc/4.0/
dc.rightsDerechos Reservados - Universidad de Santander, 2018
dc.titleAnálisis estructural y determinación de la actividad tóxica de las mutantes 8cry11l553f, 8cry11l556w, y 8cry11l553f-l556w obtenidas por mutagénesis sitio dirigida en larvas de primer estadio de aedes aegypti.
dc.typeTrabajo de grado - Pregrado


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