Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales
91 p.
- Autores:
-
Abaunza Villamizar, Sebastián Mauricio
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2018
- Institución:
- Universidad de Santander
- Repositorio:
- Repositorio Universidad de Santander
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.udes.edu.co:001/4341
- Acceso en línea:
- https://repositorio.udes.edu.co/handle/001/4341
- Palabra clave:
- Bacillus thuringiensis
Modelos heurísticos
Cry11Aa
A. aegypti
Heuristic Models
- Rights
- openAccess
- License
- Derechos Reservados - Universidad de Santander, 2018
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| dc.title.spa.fl_str_mv |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| title |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| spellingShingle |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales Bacillus thuringiensis Modelos heurísticos Cry11Aa A. aegypti Heuristic Models |
| title_short |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| title_full |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| title_fullStr |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| title_full_unstemmed |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| title_sort |
Caracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales |
| dc.creator.fl_str_mv |
Abaunza Villamizar, Sebastián Mauricio |
| dc.contributor.advisor.spa.fl_str_mv |
Suárez Barrera, Miguel Orlando |
| dc.contributor.author.spa.fl_str_mv |
Abaunza Villamizar, Sebastián Mauricio |
| dc.subject.proposal.spa.fl_str_mv |
Bacillus thuringiensis Modelos heurísticos Cry11Aa A. aegypti Heuristic Models |
| topic |
Bacillus thuringiensis Modelos heurísticos Cry11Aa A. aegypti Heuristic Models |
| description |
91 p. |
| publishDate |
2018 |
| dc.date.issued.spa.fl_str_mv |
2018-11-23 |
| dc.date.accessioned.spa.fl_str_mv |
2020-01-21T20:43:25Z |
| dc.date.available.spa.fl_str_mv |
2020-01-21T20:43:25Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Pregrado |
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http://purl.org/coar/resource_type/c_7a1f |
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spa |
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Abdullah, M. A., & Dean, D. H. (2004). Enhancement of Cry19Aa Mosquitocidal Activity against Aedes aegypti by Mutations in the Putative Loop Regions of Domain II. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Abdullah, M. A., Alzate, O., Mohammad, M., McNall, R. J., Adang, M. J., & Dean, D. H. (2003). Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Adang, M. J., Staver, M. J., Rocheleau, T. A., Leighton, J., Barker, R. F., & Thompson, D. V. (1985). Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus tkuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta . Gene. Barret, G. (2000). Chemestry and biochemestry of the amino acids. Bravo, A., Gill, S. S., & Soberón, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon. Bravo, A., Gómez, I., Porta, H., García-Gómez, B. I., Rodriguez-Almazan, C., Pardo, L., & Soberón, M. (2012). Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microbial Biotechnology. Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M. (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. ELSEVIER. Cantón, P. E., Reyes, E. Z., Escudero, I. R., Bravo, A., & Soberón, M. (2011). Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism. Peptides. Carvalho, D. O., Costa-da-Silva, A. L., Lees, R. S., & Capurro, M. L. (2013). Two step male release strategy using transgenic mosquito lines to control transmission of vector-borne diseases. Acta Topica. Craveiroa, K. I., Júnior, J. E., Silvaa, M. C., Macedo, L. L., Lucena, W. A., Silva, M. S., . . . Santiago, A. D. (2010). Variant Cry1Ia toxins generated by DNA shuffling are active against sugarcane giant borer. Journal of Biotechnology. Dean, Rajamohan, Lee, Wu, Chen, Alcantara, & Hussain. (1996). Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis a minireview. Gene. Deist, B. R., Rausch, M. A., Fernandez-Luna, M. T., Adang, M. J., & Bonning, B. C. (2014). Bt Toxin Modification for Enhanced Efficacy. Toxins. Delécluse, A., Rosso, M.-L., Ragni, & Adriano. (1995). Cloning and Expression of a Novel Toxin Gene from Bacillus thuringiensis subsp. jegathesan Encoding a Highly Mosquitocidal Protein. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Didelot, X., Barker, M., Falush, D., & Priest, F. G. (2009). Evolution of pathogenicity in the Bacillus cereus group. ELSEVIER. DONOVAN, W. P., DANKOCSIK, C., & GILBERT, M. P. (1988). Molecular Characterization of a Gene Encoding a 72-Kilodalton Mosquito-Toxic Crystal Protein from Bacillus thuringiensis subsp. israelensis. Joournal of Bacteriology . Fernández, L. E., Pérez, C., Segovia, L., Rodríguez, M. H., Gill, S. S., Bravo, A., & Soberón, M. (2005). Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop a-8 of domain II. FEBS. Girard, F., Vachon, V., Préfontaine, G., Marceau, L., Schwartz, J.-L., Masson, L., & Laprade1, R. (2009). Helix 4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Grochulski, P., Masson, L., Borisova, S., Pusztai-Carey, M., Schwartz, J.-L., Brousseau, R., & Cygler, M. (1995). Bacillus thuringiensis CrylA(a) Insecticidal Toxin: Crystal Structure and Channel Formation. JMB. Hofte, H., & Whiteley, H. R. (1989). Insecticidal Crystal Proteins of Bacilllus thuringiensis. MICROBIOLOGICAL REVIEWS,. HOFTE, H., GREVE, H. d., SEURINCK, J., JANSENS, S., MAHILLON, J., AMPE, C., . . . VAECK, M. (1986). Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. ISAAA. (2011). Global Status of Commercialized Biotech/GM Crops. International Service For The Acquisition Of Agro-Biotech Applications. Lecadet, M., Frachon, E., Dumanoir, V. C., Ripouteau, H., Hamon, S., Laurent, P., & Thiéry, I. (1999). Updaiting the H-antigen classification of Bacillus thurigiensis. Journal of Applied Microbiology. Lee, S., Aimanova, K., & Gill, S. (2014). Alkaline phophatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti. Insect Biochemistry and Molecular Biology, 54, 112-121. Li, J., Caroll, J., & Ellar, D. (1991). Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution. Nature. Li, S., Wang, Z., Zhou, Y., Li, C., Wang, G., Wang, H., . . . Lang, Z. (2018). Expression of cry2Ah1 and two domain II mutants in transgenic tobacco confers high resistance to susceptible and Cry1Ac-resistant cotton bollworm. SCIENTIFIC REPORTS. Likitvivatanavong, S., Chen, J., Evans, A. E., Bravo, A., Soberón, M., & Gill, S. S. (2011). Multiple receptors as targets of Cry toxins ins mosquitoes. Agric Food Chem . Liu, X. S., & H.Dean, D. (2006). Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin. Protein Engineering, Design & Selection. liu, Y., Wang, Q., Wang, F., Ding, X., & Lia, L. (2010). Residue 544 in domain III of the Bacillus thuringiensis Cry1Ac toxin is involved in protein structure stability. Protein Journal. López-Pazos, S. A., & Cerón, J. (2010). Proteínas Cry de Bacillus thuringiensis y su interacción con coleópteros. Lucena, W. A., Pelegrini, P. B., Martins-de-Sa, D., Fonseca, F. C., Jr., J. E., Macedo, L. L., . . . Grossi-de-Sa, M. F. (2014). Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins. Toxins. Maagd, R. A., Bravo, A., Berry, C., Crickmore, N., & Schnepf, H. E. (2003). STRUCTURE, DIVERSITY, AND EVOLUTION OF PROTEIN TOXINS FROM SPORE-FORMING ENTOMOPATHOGENIC BACTERIA. Genet. Mandal, C. C., Gayen1, S., & Basu, A. (2007). Prediction-based protein engineering of domain I of Cry2A entomocidal toxin of Bacillus thuringiensis for the enhancement of toxicity against lepidopteran insects. Protein Engineering, Design & Selection. Melo, A. L., Soccol, V. T., & Soccol, C. R. (2014). Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review. Biotechnol. OMS. (28 de Marzo de 2018). Organización Mundial de la Salud. Orduz, S., Realpe, M., Arango, R., Murillo, L. Á., & Delécluse, A. (1998). Sequence of the cry11Bb gene from Bacillus thruingiensis subsp. medellin and toxicity analysis of its encoded protein . Biochimica et Biophysica acta. Palma, L., Muñoz, D., Berry, C., Murillo, J., & Caballero, P. (2014). Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity. Toxins. Palma, L., Muñoz, D., Berry, C., Murillo, J., & Caballero, P. (2014). Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity. Toxins. Palma, L., Muñoz, D., Berry, C., Murillo, J., & Caballero, P. (2014). Bacillus thuringiensis toxins: an overview of their biocidal activity . Toxins. Pao-intara, M., Angsuthanasombat, C., & Panyim, S. (1988). The mosquito larvicidal activity of 130 kDa delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment. Biochem Biophys Res Commun. Parra, J. L. (2016). Mutación Sitio Dirigida de las posiciones 553F y 556W de la variante 8Cry11 de Bacillus thuringiensis. Peterson, B., Bezuidenhout, C. C., & Berg, J. V. (2017). An Overview of Mechanisms of Cry Toxin Resistance in Lepidopteran Insects. Biological and Microbial Control. Pigott, C. R., & Ellar, D. J. (2007). Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS. Rajamohan, F., Alzate, O., Cotrill, J. A., Curtiss, A., & Dean, D. (1996). Protein engineering of Bacillus thuringiensis d-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae. Proc. Natl. Acad. Sci. Ruiz, L. M., Segura, C., Trujillo, J., & Orduz, S. (2004). In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae). Mem Inst Oswaldo Cruz. Sanahuja, G., Banakar, R., Twyman, R. M., Capell, T., & Christou, P. (2011). Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechonology Journal. Sansinenea, E. (2012). Discovery and Description of Bacillus thuringiensis. En E. Sansinenea, Bacillus thuringiensis biotechnology (págs. 3-18). Sauka, D., Monella, R., & Benintende, G. (2010). Detection of the mosquitocidal toxin genes enconding Cry11 proteins from Bacillus thuringiensis using a novel PCR-RFLP method. Revista Argentina de Microbiología, 23-26. Schnepf, E., Crickmore, N., Rie, J. V., Lereclus, D., Baum, J., Feitelson, J., . . . Dean, D. H. (1998). Bacillus thuringiensis and Its Pesticidal Crystal Proteins. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS. Soberón, M., & Bravo, A. (2007). Las toxinas Cry de Bacillus thurigiensis: modo de acción y consecuencias de su aplicación . Biotecnología . Soberón, Pardo-Lopez, Lopez, Gómez, Tabashnik, & Bravo, A. (2007). Engineering. Science. Tanaka, S., Endo, H., Adegawa, S., Kikuta, S., & Sato, R. (2016). Functional characterization of Bacillus thuringiensis Cry toxin receptors explains resistance in insects. The FEBS journal. Wedge, D. C., Rowe, W., Kell, D. B., & Knowles, J. (2009). In silico modelling of directed evolution: Implications for experimental design and stepwise evolution. Journal of Theoretical Biology. Wirth, M., Walton, W., & Federici, B. (2015). Evolution of Resistance in Culex quinquefasciatus (Say) Selected With a Recombinant Bacillus thuringiensis Strain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, From Lysinibacillus sphaericus. J Med Entomol 52(5), 1028-1035. Wu, S., Koller, S., Miller, D., Bauer, L., & Dean, D. (2000). Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop. FEBS. Xu, C., Wang, B.-C., Yu, Z., & Sun, M. (2014). Structural Insights into Bacillus thuringiensis Cry,Cyt and Parasporin Toxins . Toxins. Xu, Y., Nagai, M., Bagdasarian, M., Smith, T. W., & Walker, E. D. (2001). Expression of the p20 Gene from Bacillus thuringiensis H-14 Increases Cry11A Toxin Production and Enhances MosquitoLarvicidal Activity in Recombinant Gram-Negative Bacteria. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. YAMAGIWA, M., SAKAGAWA, K., & SAKAI, H. (2014). Functional Analysis of Two Processed Fragments of Bacillus thruingiensis Cry11A toxin. Bioscience, Biotechnology, and Biochemistry. Zhang, Q., Hua, G., Bayyareddy, K., & Adang, M. (2013). Analyses of a-amylase and a-glucosidase in the malaria vector mosquito, Anopheles gambiae, as receptors of Cry11Ba toxin of Bacillus. Insect Biochemistry and Molecular Biology (43)10, 907–915. Zhang, X., Candas, M., Griko, N. B., Taussing, R., & Bulla, L. A. (2006). Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells. PNAS. Zouari, N., Dhouib, A., Ellouz, R., & Jaoua, S. (1998). Nutritional requirements of a strain of bacillus thuringiensis subsp. kurstaki and use of gruel hydrolysate for the formulation of a new medium for δ-endotoxin production. Applied Biochemistry and Biotechnology, 41-52. |
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Derechos Reservados - Universidad de Santander, 2018 |
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Bucaramanga : Universidad de Santander, 2018 |
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Suárez Barrera, Miguel Orlando335becb1-2371-418a-acf8-21265141ac00-1Abaunza Villamizar, Sebastián Mauriciocae1bd50-1b8c-4969-bf01-bd079c302746-12020-01-21T20:43:25Z2020-01-21T20:43:25Z2018-11-2391 p.Bacillus 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.Bacillus 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.PregradoMicrobiólogo Industrial1. 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 .............................................................................. 83Ej. 1application/pdfT 33.18 A118chttps://repositorio.udes.edu.co/handle/001/4341spaBucaramanga : Universidad de Santander, 2018Facultad de Ciencias Exactas, Naturales y AgropecuariasMicrobiología IndustrialAbdullah, M. A., & Dean, D. H. (2004). Enhancement of Cry19Aa Mosquitocidal Activity against Aedes aegypti by Mutations in the Putative Loop Regions of Domain II. APPLIED AND ENVIRONMENTAL MICROBIOLOGY.Abdullah, M. A., Alzate, O., Mohammad, M., McNall, R. J., Adang, M. J., & Dean, D. H. (2003). Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering. APPLIED AND ENVIRONMENTAL MICROBIOLOGY.Adang, M. J., Staver, M. J., Rocheleau, T. A., Leighton, J., Barker, R. 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