Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620
Digital
- Autores:
-
Sánchez-Ballesteros, Laura Milena
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2021
- Institución:
- Universidad de Santander
- Repositorio:
- Repositorio Universidad de Santander
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.udes.edu.co:001/5528
- Acceso en línea:
- https://repositorio.udes.edu.co/handle/001/5528
- Palabra clave:
- Bacillus thuringiensis
Parasporins
Site-Directed mutagenesis
Parasporinas
Mutagénesis sitio-dirigida
- Rights
- openAccess
- License
- Derechos Reservados - Universidad de Santander, 2021
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Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| title |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| spellingShingle |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 Bacillus thuringiensis Parasporins Site-Directed mutagenesis Parasporinas Mutagénesis sitio-dirigida |
| title_short |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| title_full |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| title_fullStr |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| title_full_unstemmed |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| title_sort |
Determinación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620 |
| dc.creator.fl_str_mv |
Sánchez-Ballesteros, Laura Milena |
| dc.contributor.advisor.none.fl_str_mv |
Suárez-Barrera, Miguel Orlando Rueda-Forero, Nohora Juliana |
| dc.contributor.author.none.fl_str_mv |
Sánchez-Ballesteros, Laura Milena |
| dc.subject.proposal.eng.fl_str_mv |
Bacillus thuringiensis Parasporins Site-Directed mutagenesis |
| topic |
Bacillus thuringiensis Parasporins Site-Directed mutagenesis Parasporinas Mutagénesis sitio-dirigida |
| dc.subject.proposal.spa.fl_str_mv |
Parasporinas Mutagénesis sitio-dirigida |
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Digital |
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2021 |
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2021-08-20T20:25:06Z |
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2021-08-20T20:25:06Z |
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2021-01-28 |
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Trabajo de grado - Pregrado |
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spa |
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Derechos Reservados - Universidad de Santander, 2021 |
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Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0) |
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Derechos Reservados - Universidad de Santander, 2021 Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0) https://creativecommons.org/licenses/by-nc-nd/4.0/ http://purl.org/coar/access_right/c_abf2 |
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openAccess |
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85 p |
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application/pdf |
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Bucaramanga : Universidad de Santander, 2021 |
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Facultad de Ciencias Exactas, Naturales y Agropecuarias |
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Bucaramanga, Colombia |
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Microbiología Industrial |
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Universidad de Santander |
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Suárez-Barrera, Miguel Orlando335becb1-2371-418a-acf8-21265141ac00Rueda-Forero, Nohora Julianafdefebdb-5a74-4c59-ba33-4ff4cc09bca4Sánchez-Ballesteros, Laura Milena11ac3a08-9050-4fa4-854b-0675d12b00372021-08-20T20:25:06Z2021-08-20T20:25:06Z2021-01-28DigitalBacillus thuringiensis es una bacteria Gram positiva estudiada por la producción de cristales parasporales con potencial insecticida. Recientemente, se descubrió un nuevo grupo de proteínas cristal, denominadas como parasporinas. Dentro de este nuevo grupo, se destaca la parasporina 2(PS2Aa1), que se caracteriza por exhibir efectos citotóxicos frente a diversas líneas celulares de cáncer de colon, estableciéndose como una posible alternativa a los tratamientos tradicionales para combatir esta enfermedad. Sin embargo, actualmente no se encuentra bien definido su mecanismo de acción ni los receptores involucrados. Con la actual investigación se emplearon tecnologías de mutagénesis sitio-dirigidas, con el fin de obtener variantes de PS2Aa1 mejoradas. Para ello, primero se realizó un estudio in silicio de la proteína, para buscar secuencias homologas y dominios conservados, y llevar a cabo el diseño de los primers. Luego se realizó transformación para incorporar el gen de interés con el vector pET30a a las células DE3BL21. Se llevaron a cabo los ensayos de mutagénesis sitio-dirigida asistida con PCR para obtener las mutantes a partir de PS2Aa1; a partir de la biblioteca producida, se hicieron ensayos de citotoxicidad por medio del Kit Sulforhodamine B Cell Cytoxicity Assay en células de cáncer de colon SW-480 y SW-620. A partir de estos, se pudo determinar que PS2Aa1 poseía dominios conservados de familias de proteínas β-PFT de tipo aerolisina. A partir de la mutagénesis se logró obtener una librería de numerosas mutantes, en donde solo 6 presentaron las mutaciones de interés. En los ensayos de citotoxicidad, se evidenciaron 4 mutantes que inhibieron el crecimiento de las células cancerígenas. Sin embargo, las mutantes presentaron inhibición de crecimiento en las células CHO-K1, aunque menor con respecto a la nativa, por lo cual es necesario llevar a cabo más estudios con el fin de establecer si dichos cambios fueron significativos en la estructura-función de la proteína.Bacillus thuringiensis is a Gram-positive bacterium studied for the production of parasporal crystals with insecticide potential. Recently, a new group of crystal proteins, called parasporins, was discovered. Within this new group, parasporin 2 (PS2Aa1) stands out, which is characterized by displaying cytotoxic effects against various colon cancer cell lines, establishing itself as a possible alternative to traditional treatments to combat this disease. However, its mechanism of action and the recipients involved are currently not well defined. Current research used site-directed mutagenesis technologies to obtain improved PS2Aa1 variants. To do this, we first conducted an-in silicon study of the protein, to look for homologous sequences and conserved domains, and to carry out the design of primers. Transformation was then performed to incorporate the gene of interest with the pET30a vector into the DE3BL21 cells. PCR-assisted site-directed mutagenesis assays were conducted to obtain mutants from PS2Aa1; from the produced library, cytotoxicity assays using the Sulforhodamine B Cell Cytoxicity Assay Kit were performed on colon cancer cells SW-480 and SW-620. From these, it was possible to determine that PS2Aa1 possessed conserved domains of family of aerolysine-type β-PFT proteins. From the mutagenesis it was possible to obtain a library of numerous mutants, where only 6 presented mutations of interest. In cytotoxicity trials, 4 mutants were shown to inhibit the growth of cancer cells. However, mutants presented growth inhibition in CHO-K1 cells, although less than native, so it is necessary to carry out more studies in order to establish if such changes were significant in the structure-function of the protein.PregradoMicrobiólogo Industrial1 ed.Resumen ..................................................................................................................................11 Abstract.....................................................................................................................................13 Introducción ..............................................................................................................................15 1. Planteamiento del Problema ..............................................................................................19 2. Justificación .......................................................................................................................21 3. Pregunta de Investigación ..................................................................................................24 4. Hipótesis ............................................................................................................................25 4.1. Hipótesis de Investigación. ..................................................................................25 4.2. Hipótesis Nula. .....................................................................................................25 4.3. Hipótesis Alternativa. ...........................................................................................25 5. Objetivos ............................................................................................................................26 5.1. Objetivo General. .................................................................................................26 5.2. Objetivos Específicos. ..........................................................................................26 6. Marco Referencial ..............................................................................................................27 6.1. Generalidades de Bacillus thuringiensis (Bt) ........................................................27 6.2. Parasporinas........................................................................................................29 6.3. Parasporina 2Aa1 ................................................................................................31 6.4. Estructura de la PS2Aa1 ......................................................................................32 6.5. Mecanismo de Acción de la PS2Aa1 ...................................................................34 6.6. Técnica de Mutagénesis Sitio-Dirigida para el Mejoramiento de Proteínas ..........37 7. Marco Legal .......................................................................................................................40 8. Método ...............................................................................................................................41 8.1. Diseño del Estudio: ..............................................................................................41 8.2. Metodología .........................................................................................................41 8.2.1.Identificación In Sillico de las Regiones de Interés para el Diseño de Primers. 41 8.2.2.Diseño de Primers para la Mutagénesis. .....................................................41 8.2.3.Cepas Bacterianas y Condiciones de Cultivo ..............................................42 8.2.4.Transformación del Plásmido pET30A+PS2Aa1 en Células de E. coli DE3BL21. ..........................................................................................................................42 8.2.5.Extracción de ADN Plasmídico y Ensayos de Restricción de pET30a-PS2Aa1. 43 8.2.6.Ensayos de Mutagénesis por Medio del Kit GeneArt Site-Directed Mutagenesis Plus. ..............................................................................................................44 8.2.7.Extracción de ADN Plasmídico de las Mutantes de PS2Aa1 y Cuantificación. 46 8.2.8.Extracción de Proteínas y Cuantificación por Medio de Método de Bradford 46 8.2.9.Tratamientos con Proteínasa K para la Activación de las Mutantes. ...........47 8.3.Líneas Celulares ............................................................................................47 8.3.1.Ensayos de Citotoxicidad de las Mutantes de PS2Aa1 en Células de Cáncer de Colon SW-480 y SW-620. .............................................................................................47 8.3.2.Análisis Estadístico de los Datos.................................................................48 9. Resultados .........................................................................................................................49 10. Discusión ...........................................................................................................................65 11. Conclusiones .....................................................................................................................72 12. Recomendaciones .............................................................................................................74 Referencias Bibliográficas .........................................................................................................75 Apéndices .................................................................................................................................8585 papplication/pdfT 33.21 S162dhttps://repositorio.udes.edu.co/handle/001/5528spaBucaramanga : Universidad de Santander, 2021Facultad de Ciencias Exactas, Naturales y AgropecuariasBucaramanga, ColombiaMicrobiología IndustrialDerechos Reservados - Universidad de Santander, 2021info:eu-repo/semantics/openAccessAtribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)https://creativecommons.org/licenses/by-nc-nd/4.0/http://purl.org/coar/access_right/c_abf2Bacillus thuringiensisParasporinsSite-Directed mutagenesisParasporinasMutagénesis sitio-dirigidaDeterminación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620Trabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fTextinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPhttp://purl.org/coar/version/c_71e4c1898caa6e32Todas las AudienciasAbcam. (2018). Sulforhodamine B Cell Cytotoxicity Assay Kit. 408, 2. http://www.biovision.com/documentation/datasheets/K943.pdf.Abe, Yuich, Inoue, H., Ashida, H., Maeda, Y., Kinoshita, T., & Kitada, S. (2017). Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547. Journal of Invertebrate Pathology, 142, 71–81. https://doi.org/10.1016/j.jip.2016.11.008.Abe, Yuichi, Shimada, H., & Kitada, S. (2008). Raft-targeting and oligomerization of parasporin-2, a Bacillus thuringiensis crystal protein with anti-tumour activity. Journal of Biochemistry, 143(2), 269–275. https://doi.org/10.1093/jb/mvm220.Akiba, T., Abe, Y., Kitada, S., Kusaka, Y., Ito, A., Ichimatsu, T., Katayama, H., Akao, T., Higuchi, K., Mizuki, E., Ohba, M., Kanai, R., & Harata, K. (2004). Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cells. Acta Crystallographica Section D: Biological Crystallography, 60(12 II), 2355–2357. https://doi.org/10.1107/S0907444904026307.Akiba, T., Mizuki, E., Katayama, H., Higuchi, K., Kusaka, Y., Abe, Y., Harata, K., Ohba, M., Ichimatsu, T., Ito, A., Kitada, S., Kanai, R., & Akao, T. (2009). Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells. Journal of Molecular Biology, 386(1), 121–133. https://doi.org/10.1016/j.jmb.2008.12.002.Akiba, T., & Okumura, S. (2017). Parasporins 1 and 2: Their structure and activity. Journal of Invertebrate Pathology, 142(October), 44–49. https://doi.org/10.1016/j.jip.2016.10.005Aktar, N., Karim, M. M., Khan, S., & Begum, A. (2019). In silico Studies of Parasporin Proteins : Structural and Functional Insights and Proposed Cancer Cell Killing Mechanism for Parasporin 5 and 6. May. https://doi.org/10.25163/microbbioacts.21007A0621280219Al-hilu, S. A. (2020). Dual Role of Bacteria in Carcinoma : Stimulation and Inhibition. 2020.Aldeewan, A., & Zhang, Y. (2016). Bacillus thuringiensis Parasporins Functions on Cancer Cells Bacillus thuringiensis Parasporins Functions on Cancer Cells. International Journal of Pure & Applied Bioscience, 2(4), 67–74.Assaeedi, A. S., & Osman, G. H. (2017). Isolation, Cloning, DNA Sequencing and Bioinformatics Analysis of the Parasporin–1 Gene of Bacillus thuringiensis. Journal of Proteomics & Bioinformatics, 10(5). https://doi.org/10.4172/jpb.1000435Bachman, J. (2013). Site-directed mutagenesis. In Methods in Enzymology (1st ed., Vol. 529). Elsevier Inc. https://doi.org/10.1016/B978-0-12-418687-3.00019-7Baindara, P., & Mandal, S. M. (2020). Biochimie Bacteria and bacterial anticancer agents as a promising alternative for cancer therapeutics. Biochimie, 177, 164–189. https://doi.org/10.1016/j.biochi.2020.07.020Balabanova, L., Golotin, V., Podvolotskaya, A., & Rasskazov, V. (2015). 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S., Lukoyanova, N., Robertson, S., Moss, D. S., Mcclane, B. A., & Basak, A. K. (2011). Structure of the Food-Poisoning Clostridium perfringens Enterotoxin Reveals Similarity to the Aerolysin-Like Pore-Forming Toxins. Journal of Molecular Biology, 413(1), 138–149. https://doi.org/10.1016/j.jmb.2011.07.066Castañet-Martínez, C. E., & Moreno-reyes, S. (2016). Bacillus thurinngiennsis : Características y uso en el control de Aedes aegypti.Chakrabarty, A. M., Bernardes, N., & Fialho, A. M. (2014). Bacterial proteins and peptides in cancer therapy. Bioengineered, 5(4), 234–242. https://doi.org/10.4161/bioe.29266Chubicka, T., Girija, D., Deepa, K., Salini, S., Meera, N., Raghavamenon, A. C., Divya, M. K., & Babu, T. D. (2018). A parasporin from Bacillus thuringiensis native to Peninsular India induces apoptosis in cancer cells through intrinsic pathway. Journal of Biosciences, 43(2), 407–416. https://doi.org/10.1007/s12038-018-9759-0Cirauqui, N., Abriata, L. A., Van Der Goot, F. G., & Dal Peraro, M. (2017). Structural, physicochemical and dynamic features conserved within the aerolysin pore-forming toxin family. Scientific Reports, 7(1), 1–12. https://doi.org/10.1038/s41598-017-13714-4Crickmore, N., Berry, C., Panneerselvam, S., Mishra, R., Connor, T. R., & Bonning, B. C. (2020). A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. Journal of Invertebrate Pathology, July, 107438. https://doi.org/10.1016/j.jip.2020.107438De, E., & Min, T. (2019). Medio SOC Test microbiológico. 5–6.Domanska, B. (2016). Mode of action of a human cancer cell active toxin (Parasporin-3) from Bacillus thuringiensis. 318. https://doi.org/http://sro.sussex.ac.uk/66159/1/Domanska,%20Barbara.pdfDubey, K. K., Pramanik, A., Ankush, Khushboo, & Yadav, J. (2019). Enzyme engineering. Biomass, Biofuels, Biochemicals: Advances in Enzyme Technology, 325–347. https://doi.org/10.1016/B978-0-444-64114-4.00012-1Edelheit, O., Hanukoglu, A., & Hanukoglu, I. (2009). Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnology, 9, 1–8. https://doi.org/10.1186/1472-6750-9-61Ekino, K., Okumura, S., Ishikawa, T., Kitada, S., Saitoh, H., Akao, T., Oka, T., Nomura, Y., Ohba, M., Shin, T., & Mizuki, E. (2014). Cloning and characterization of a unique cytotoxic protein parasporin-5 produced by Bacillus thuringiensis a1100 strain. Toxins, 6(6), 1882–1895. https://doi.org/10.3390/toxins6061882Elsevier. (2019). Muerte celular: apoptosis, necrosis y autofagia. Elsevier. https://www.elsevier.com/es-es/connect/medicina/bioquimica-muerte-celular-apoptosis-necrosis-y-autofagiaEngland, P. H. (2017). Cell line profile CHO K1 ECACC. European Collection of Authenticated Cell Cultures, 2780(93112519), 1–3. https://www.phe-culturecollections.org.uk/media/133182/mda-mb-231-cell-line-profile.pdfFederici, Brian A., Park, Hyun-Woo & Bideshi, D. K. (2013). Overview of the Basic Biology of Bacillus thuringiensis with Emphasis on Genetic Engineering of Bacterial Larvicides for Mosquito Control. The Open Toxinology Journal, 3(1), 83–100. https://doi.org/10.2174/1875414701003010083Ge, A. Z., Rivers, D., Milne, R., & Dean, D. H. (1991). Functional domains of Bacillus thuringiensis insecticidal crystal proteins: Refinement of heliothis virescens and Trichoplusia ni specificity domains on CryIA(c). Journal of Biological Chemistry, 266(27), 17954–17958. https://doi.org/10.1016/s0021-9258(18)55221-2GeneArt ® Site-Directed Mutagenesis PLUS System. (2013). 3–5.Globocan. (2019). Colorectal cancer. 876, 1–2.Globocan. (2020). 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