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Descripción
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| A number of microorganisms are being studied as potential producers of H2 through biophotolysis, indirect biophotolysis, photo-fermentations or dark-fermentations. Microorganisms produce H2 by the activity of either hydrogenases or nitrogenases. Hydrogenases catalyze the reaction: 2H+ + 2e- ? H2, whereas nitrogenases catalyze the reduction of N2 with the following limiting stoichiometry: N2 + 8H+ + 8e- ? H2 + 2NH3. In this work, we have coordinated aspects of both pathways to develop optimized biocatalysts for H2 production using two genetic modules: 1. Engineering an H2 responsive genetic circuit in the purple non-sulphur N2-fixing bacterium Rhodobacter capsulatus SB1003. R. capsulatus carries a system to detect H2 that is composed of three proteins: a H2-sensor hydrogenase (HupUV), a histidine kinase (HupT) and a response regulator HupR (Vignais et al., 2005). In the presence of H2, this sensor triggers expression of hydrogenase structural and biosynthetic genes. Taking advantage of this system, we have introduced a reporter gene under the control of hupS promoter and removed the uptake hydrogenase, generating a new biological-sensor strain capable of producing a measurable and proportional signal when H2 is present in the cell. 2. Generating in vitro evolution of the molybdenum nitrogenase structural genes nifH, nifD and nifK by random mutagenesis. The resulting variants were cloned under nifH promoter control into a broad-host-range vector (Kovach et al., 1995) optimized for diazotrophic conditions. Libraries obtained (around 4 x 106 clones) were introduced and expressed in the strain carrying the modified biological hydrogen sensor. The combination of both tools results in the development of a genetic circuit for the highthroughput screening of H2 overproducing nitrogenase variants thus allowing detection and isolation of clones that present a significant signal increased, through the use of cellsorting cytometry. Thus far, around 1500 clones have been successfully selected by this method; and several of them were able to produce until 18 times more H2 than the control strain. This methodology has potential to be implemented in other biological systems to increase H2 production yields. | |
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Internacional
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No |
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Nombre congreso
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XI Reunión de Microbiología Molecular |
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Tipo de participación
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960 |
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Lugar del congreso
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Sevilla |
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Revisores
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Si |
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ISBN o ISSN
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0000000000 |
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DOI
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Fecha inicio congreso
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06/09/2016 |
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Fecha fin congreso
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08/09/2016 |
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Desde la página
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12 |
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Hasta la página
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12 |
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Título de las actas
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XI Reunión de Microbiología Molecular |