Examinando por Materia "Bacillus thuringiensis"
Mostrando 1 - 12 de 12
Resultados por página
Opciones de clasificación
- PublicaciónAcceso abiertoAnálisis 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.(Bucaramanga : Universidad de Santander, 2018, 2018-11-23) Herrera Pineda, Diego Fernando; Suárez Barrera, Miguel Orlando; Rueda Forero, Nohora JulianaBacillus 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.
- PublicaciónAcceso abiertoCaracterizaci ón molecular de genes cry1, cry2, cry3 y cry4 en aislados de Bacillus thuringiensis y determinación de su actividad bioinsecticida en larvas de Aedes aegypti(2013-02-20) Galvis Serrano, Nestor FabiánChemical insecticides can be toxic and cause environmental degradation. Therefore, biological control of insects represents an alternative of low ecological impact. Bacillus thuringiensis is a spore-forming Gram-positive bacterium that produces parasporal crystals of a proteic nature, formed by delta endotoxins that are toxic to a large number of insects and are biodegradable and innocuous to other species. In the present work 13 native strains of B. thuringiensis were isolated from soil samples and identified by selective methods and the BBL CRYSTAL method. In the molecular characterization utilizing specific primers for the identification of cry1, cry2, cry3 y cry4 genes, eight isolates presented the cry3 gene and two presented the cry2 gene. These two latter isolates were used in a bioassay on Aedes aegypti larvae to determine their toxic effect, showing that the preliminary toxicity essay of the BtUDES2 isolate presented a lethality of 56.67%. When determining the lethal concentration of this same isolate, an average lethal concentration of 11.4333ng·ml-1 and a total lethal concentration of 17.1542ng·ml-1 were found.
- PublicaciónAcceso abiertoCaracterización de nuevas proteínas Cry11 obtenidas por modelos heurísticos computacionales(Bucaramanga : Universidad de Santander, 2018, 2018-11-23) Abaunza Villamizar, Sebastián Mauricio; Suárez Barrera, Miguel OrlandoBacillus 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.
- PublicaciónAcceso abiertoCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity(2011-10) Hussain, Syed-Rehan A.; Florez, Alvaro M.; Osorio, Cristina; Dean, Donald H.; Alzate, OscarThe centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.
- PublicaciónAcceso abiertoDeterminación del Potencial de Citotoxicidad de Parasporinas tipo PS2Aa1 Obtenidas por Evolución Dirigida, en Células de Cáncer de Colon SW-480 y SW-620(Bucaramanga : Universidad de Santander, 2021, 2021-01-28) Sánchez-Ballesteros, Laura Milena; Suárez-Barrera, Miguel Orlando; Rueda-Forero, Nohora JulianaBacillus 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.
- PublicaciónAcceso abiertoDNA secondary structure formation by DNA shuffling of the conserved domains of the Cry protein of Bacillus thuringiensis(2017-12) Pinzón Reyes, Efraín-Hernando; Sierra, Daniel A.; Suárez Barrera, Miguel Orlando; Orduz, Sergio; Florez, Alvaro M.Background The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy (ΔG¯¯¯¯¯¯¯¯) were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G. Results The results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA. Conclusion These results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.
- PublicaciónAcceso abiertoEvaluación in Vitro de Nuevas Parasporinas Obtenidas por Evolución Dirigida en Células Cancerígenas de Colon SW480 y SW620(Universidad de Santander, 2023-11-15) Ardila-Carreño, Natalia Andrea; Alarcón-Aldaba, Juan Sebastián; Rueda-Forero, Nohara Juliana; Suárez -Barrera, Miguel Orlando; Suárez -Barrera, Miguel Orlando; Farfan-Garcia, Ana Elvira; Valdivieso-Quintero, Wilfredo; Biología Molecular y BiotecnologíaIntroducción: Los tratamientos actuales contra el cáncer colorrectal son la radioterapia y la quimioterapia, sin embargo, presentan efectos secundarios debido a su poca especificidad. Por eso, se buscan nuevas alternativas terapéuticas para reemplazarlos, entre estas, surgen moléculas obtenidas de bacterias, como las parasporinas, más específicamente la parasporina PS2Aa1 (Mpp46Aa1). Este estudio tiene como finalidad determinar la actividad citotóxica de proteínas mutantes de PS2Aa1 obtenidas por aproximación dirigida al sitio en líneas celulares de cáncer colorrectal SW480 y SW620. Materiales y métodos: Las proteínas se obtuvieron por medio del Laboratorio de Biología Molecular de la Universidad de Santander. Las parasporinas tipo PS2Aa1 fueron utilizadas contra las líneas celulares en los ensayos de citotoxicidad, índices de selectividad, y activación de caspasas 3/7. Resultados: La mutante N65 con las sustituciones K92R (dominio I), L175D (dominio II) y S218G (dominio III) y la mutante 3-35 con una sustitución en G257V (dominio I) presentaron un IC50 de 1.0-1.4 y 1.2-1.7 μg/mL comparadas con la PS2Aa1 con un IC50 1.75-2.12 y 1.75-2.12 μg/mL, mientras que la sustitución de G256A de la 0-15 obtuvo un desempeño similar a la PS2Aa1, frente a las líneas celulares cancerígenas SW480 y SW620. Discusión: Las sustituciones en PS2Aa1, produjeron cambios conformacionales que potenciaron o no la actividad citotóxica e inducción de caspasas 3/7 como se ha propuesto en la literatura. Conclusiones: Las modificaciones de la PS2Aa1 revelan la utilidad de la mutación por evolución dirigida en la creación de variantes que potencian la actividad citotóxica frente a líneas celulares de cáncer de colon, manteniendo o aumentando su selectividad frente a líneas celulares de cáncer de colon.
- PublicaciónAcceso abiertoGeneration of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling(2020-07-27) Pinzón-Reyes, Efraín Hernando; Sierra-Bueno, Daniel Alfonso; Suarez-Barrera, Miguel Orlando; Rueda-Forero, Nohora Juliana; BiomolDirected evolution methods mimic in vitro Darwinian evolution, inducing random mutations and selective pressure in genes to obtain proteins with enhanced characteristics. These techniques are developed using trial-and-error testing at an experimental level with a high degree of uncertainty. Therefore, in silico modeling of directed evolution is required to support experimental assays. Several in silico approaches have reproduced directed evolution, using statistical, thermodynamic, and kinetic models in an attempt to recreate experimental conditions. Likewise, optimization techniques using heuristic models have been used to understand and find the best scenarios of directed evolution. Our study uses an in silico model named HeurIstics DirecteD EvolutioN, which is based on a genetic algorithm designed to generate chimeric libraries from 2 parental genes, cry11Aa and cry11Ba, of Bacillus thuringiensis. These genes encode crystal-shaped δ-endotoxins with 3 conserved domains. Cry11 toxins are of biotechnological interest because they have shown to be effective as biopesticides for disease-spreading vectors. With our heuristic model, we considered experimental parameters such as DNA fragmentation length, number of generations or simulation cycles, and mutation rate, to get characteristics of Cry11 chimeric libraries such as percentage of population identity, truncation of variants obtained from the presence of internal stop codons, percentage of thermodynamic diversity, and stability of variants. Our study allowed us to focus on experimental conditions that may be useful for the design of in vitro and in silico experiments of directed evolution with Cry toxins of 3 conserved domains. Furthermore, we obtained in silico libraries of Cry11 variants, in which structural characteristics of wild Cry families were observed in a review of a sample of in silico sequences. We consider that future studies could use our in silico libraries and heuristic computational models, as the one suggested here, to support in vitro experiments of directed evolution.
- PublicaciónAcceso abiertoIdentificación y Caracterización de Parasporinas, Obtenidas de Aislados de Bacillus spp Nativos de Suelos Colombianos(Bucaramanga : Universidad de Santander, 2021, 2021-01-28) Castro Pinzón, Yaren Yorlady; Suárez Barrera, Miguel Orlando; Rueda Forero, Nohora JulianaA 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.
- PublicaciónAcceso abiertoParticipation of valine 171 in α-helix 5 of Bacillus thuringiensis Cry1Ab δ-endotoxin in translocation of toxin into Lymantria dispar midgut membranes(2010-10-01) Alzate, Oscar; Osorio, Cristina; Florez, Alvaro M.; Dean, Donald H.The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.
- PublicaciónAcceso abiertoPreferential Protection of Domains II and III of Bacillus thuringiensis Cry1Aa Toxin by Brush Border Membrane Vesicles(2010-12) Hussain, Syed-Rehan A.; Florez, Alvaro M.; Dean, Donald H.; Alzate, OscarThe surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approxima-tely 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.
- PublicaciónAcceso abiertoToxic activity, molecular modeling and docking simulations of Bacillus thuringiensis Cry11 toxin variants obtained via DNA shuffling(2018-10-17) Suárez Barrera, Miguel Orlando; Florez, Alvaro M.; Morales, Gloria M.; Rivera, Karen Viviana; Orduz, Sergio; Ochoa, Rodrigo; Guerra, Diego; Muskus, CarlosThe Cry11 family belongs to a large group of d-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an a-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes.