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Synthetic biology could harness the ability of microorganisms to use highly toxic cyanide compounds for growth applied to bioremediation of cyanide-contaminated mining wastes and areas.

La utilización de medios informáticos para procesar los contenidos curriculares de las diferentes materias, así como para aprender su propio manejo, ha crecido exponencialmente en los últimos años. El uso de las Tecnologías de la Información y la Comunicación (TIC) pueden contribuir al acceso universal a la educación mediante microproyectos de bajo coste. Sin embargo, para ello hay que diseñar y desarrollar proyectos educativos coherentes con las necesidades educativas. Con este proyecto, programas bioinformáticos básicos necesarios para el desarrollo de un proyecto de ingeniería genética se transformarán en recursos educativos con los que el alumnado no solo adquirirá nociones básicas sobre el uso y manejo de estos programas -que les serán requeridos en su vida profesional como científicos-, sino que les servirá para afianzar y contextualizar conceptos relacionados con el propio proceso. Partiendo del objetivo de la mutación de un gen concreto, el alumnado será capaz de desarrollar una estrategia que le reporte una gran cantidad de información sobre éste usando únicamente medios informáticos. The use computational toolsto process curricular contents of the different subjects, as well as to learn their own management, has grown exponentially in recent years. The use of Information and Communication Technologies (ICT) can contribute to universal access to education, equality in education, the exercise of quality teaching andto thelearning and professional development of teachers, as well as to a moreefficient management and administration of the education system. However, it is necessary to design and develop educational projects that are consistent with educational needs. With thisproject, basedon the use of basic bioinformatics programs within the framework of a genetic engineering project, students will not only acquire basic notions about the use and management of these programs, which will be required in their professional life as scientists, but also it will serve to strengthen and contextualize concepts related to the process itself. Based on the objective of mutation of a particular gene, students will be able to develop a strategy that will report a large amount of information about it using only computational tools.

Biological treatments to degrade cyanide are a powerful technology for cyanide removal from industrial wastewaters. It has been previously demonstrated that the alkaliphilic bacterium Pseudomonas pseudoalcali genes CECT5344 is able to use free cyanide and several metal − cyanide complexes as the sole nitrogen source. In this work, the strain CECT5344 has been used for detoxification of the different chemical forms of cyanide that are present in alkaline wastewaters from the jewelry industry. This liquid residue also contains large concentration s of metals like iron, copper and zinc, making this wastewater even more toxic. To elucidate the molecular mechanisms involved in the bioremediation process, a quantitative proteomic anal- ysis by LC-MS/MS has been carried out in P . pseudoalcaligene s CECT5344 cells grown with the jewelry residue as sole nitrogen source. Different proteins related to cyanide and cyanate assimilation, as well as other proteins involved in transport and resistance to metals were induced by the cyanide-cont aining jewelry residue. GntR-like regulatory proteins were also induced by this industrial residue and mutational analysis revealed that GntR-like regulatory proteins may play a role in the regulation of cyanide assimilation in P . pseudoalcaligene s CECT5344. The strain CECT5344 has been used in a batch reactor to remove at pH 9 the dif- ferent forms of cyanide present in industrial wastewaters from the jewelry industry (0.3 g/L, ca . 12 mM total cyanide, including both free cyanide and metal − cyanide complexes). This is the first report describing the biological removal at alkaline pH of such as elevated concentra- tion of cyanide present in a heterogeneou s mixture from an industrial source.

Streptococcus pneumoniae (neumococo) es un patógeno humano que puede colonizar eltracto respiratorio superior o puede causar infecciones invasivas como neumonía, meningitis yempiema, y no invasivas como otitis media, sinusitis y bronquitis, principalmente en niños,ancianos e inmunodeprimidos. Es la principal causa de muertes en niños menores de 5 años anivel mundial, sobre todo en los países en vías de desarrollo.Existen más de 90 serotipos diferentes de acuerdo a la composición de la cápsulapolisacarídica y, aunque existen vacunas comerciales basadas en la cápsula, su eficacia eslimitada y son caras. Así, tanto por la reciente aparición de cepas multirresistentes aantibióticos como por fenómenos derivados de la introducción de las vacunas polisacarídicasgracias a la gran capacidad de adaptación del neumococo es de urgente necesidaddesarrollar nuevas, más eficientes y más baratas vacunas serotipo-independientes.Esta nueva vacuna debe ser de base proteica para reducir el coste de producción, y loscandidatos, estar expuestos en superficie, antigénicamente conservados, presentes en losserotipos más prevalentes, altamente expresados, su función debe ser esencial enpatogenicidad y ser capaces de estimular repuesta celular. Con el fin de descubrir nuevoscandidatos proteicos, tanto con fines vacunales como para el desarrollo de herramientas dediagnóstico rápido, durante la presente tesis se ha desarrollado dos líneas de trabajoprincipalmente: La digestión superficial de células vivas con proteasas (1) y la producción ycaracterización de vesículas extracelulares.2. Contenido de la investigaciónEn primer lugar se llevó a cabo la optimización de la técnica del “pelado” de células vivas deneumococo con proteasas (2), tras lo cual se aplicó a una batería de aislados clínicos de adultos (3)y de niños con el fin de describir los “pan-surfomas” (conjunto de proteínas de superficie presentesen una batería de distintas estirpes), confirmando a esta técnica como una rápida y potenteaproximación para la identificación de proteínas de superficie (4, 5). Porotro lado, la reciente descripción de la producción de vesículas extracelulares en organismosGram-positivos, nos llevó a caracterizar las vesículas de neumococo debido a que dichasestructuras están enriquecidas, presuntamente, en proteínas de membrana.Así, 40 aislados clínicos (correspondientes a 29 diferentes ST) sin relaciones evolutivas, además delas estirpes de referencia R6 y TIGR4, se analizaron mediante la aproximación de ladigestión superficial de células vivas. Además, otros 4 serotipos, relacionados con diferentescapacidades de invasión (serotipos 1, 6B, 8 y 23F), se usaron para la caracterización de lasvesículas derivadas de la membrana. Todo ello permitió seleccionar 95 proteínas, cuyainmunoreactividad se probó frente a sueros de pacientes enfermos y sanos (algunas de ellas,mediantes diferentes técnicas). Esto nos permitió seleccionar un conjunto de pocasproteínas (Spr0561, Spr0247, Spr1431, Spr1754 and Sph_0062) para desarrollar un test deenfermedad neumocócica rápido, fiable, barato y fácil de usar en niños de 0->5 años, queresolvería los problemas de los métodos de diagnóstico usados hasta el momento.Por otro lado, se llevaron ensayos de supervivencia usando una estirpe virulenta del serotipo 8como agente de infección, lo cual nos permitió describir la capacidad protectora de 7 nuevasproteínas. Además, se demostró que las vesículas de membrana producidas por este mismoserotipo conferían un alto grado de protección a los ratones inmunizados..., Streptococcus pneumoniae (pneumococcus) is a human pathogen that can colonize the upperrespiratory tract and can cause invasive infections such as pneumonia, meningitis andempyema, and noninvasive as otitis media, sinusitis and bronchitis, especially in children, theelderly and immunocompromised. It is the leading cause of death in children under 5 yearsworldwide, especially in developing countries.There are more than 90 different serotypes described according to the composition of thepolysaccharide capsule and, although there are commercial capsule-based vaccines, theireffectiveness is limited and are expensive. Thus, both the recent emergence of multidrugresistantstrains to antibiotics such as phenomena arising from the introduction of thepolysaccharide vaccines, thanks to the adaptability of the pneumococcus, is an urgent need todevelop new, more efficient and cheaper vaccine serotype-independent.This new vaccine should be protein based to reduce the cost of production, and candidates,surface-exposed, antigenically conserved, presents in the most prevalent serotypes, highlyexpressed, its function must be essential for pathogenicity and capable of stimulating cellularresponse. In order to discover new protein candidates, both for vaccination and for thedevelopment of rapid diagnostic tools purposes, during this thesis two lines of work weredeveloped: The surface digestion with proteases of living (“shaving”) (1) and production andcharacterization of extracellular vesicles.Research content First, optimization of "shaving" of live pneumococcal cells with proteases (2) was carried outand then it was applied to a set of clinical isolates from adult (3) and children in order todescribe the "pan-surfome" (set of surface proteins present in a collection of different strains).This confirmed this technique as a rapid and powerful approach to surface proteinidentification (4, 5). Furthermore, the recent description of the production of extracellularvesicles in a Gram-positive bacteria, prompted us to characterize pneumococcus vesiclesbecause such structures are enriched, presumably, in membrane proteins.So, 40 clinical isolates (corresponding to 29 different ST) without evolutionary relationships, aswell as reference strains R6 and TIGR4, were analyzed by the “shaving” approach. In addition,other 4 serotypes, with different capabilities of invasion (serotypes 1, 6B, 8 and 23F) were used forthe characterization of the extracelluar vesicles (5, 6). All of this allowed to select 95 proteins andtested their immunoreactivity against sera of convalescent and healthy patients (some of them

Comunicaciones a congresos

Comunicaciones a congresos

Comunicaciones a congresos

Comunicaciones a congresos

Surface proteins play key roles in the interaction between cells and their environment, and in pathogenic microorganisms they are the best targets for drug or vaccine discovery and/or development. In addition, surface proteins can be the basis for serodiagnostic tools aiming at developing more affordable techniques for early diagnosis of infection in patients. We carried out a proteomic analysis of a collection of pediatric clinical isolates of Streptococcus pneumoniae, an important human pathogen responsible for more than 1.5 million child deaths worldwide. For that, cultured live bacterial cells were shaved with trypsin, and the recovered peptides were analyzed by LC/MS/MS. We selected 95 proteins to be produced as recombinant polypeptides, and printed them on an array. We probed the protein array with a collection of patient sera to define serodiagnostic antigens. The mass spectrometry proteomics data correspond to those published in [1] and have been deposited to the ProteomeXchange Consortium [2] via the PRIDE partner repository [3] with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD001740. The protein array raw data are provided as supplemental material in this article. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license