Monitoring of Gene Expression in Bacteria during Infections Using an Adaptable Set of Bioluminescent, Fluorescent and Colorigenic Fusion Vectors

A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfpmut3.1, amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process

The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

Temperature microsensor/microactuator based on magnetic microwire for MEMS applications

The aim of this paper has been the development of a new type of temperature microsensor/microactuator working on the principle of the thermo-elastic (TE) deformation of multilayer magnetic microwire consisting of a glass-coated CoSiB metallic core and an electroplated CoNi external shell. The application of an electrical current along the microwire in the range 20-35 mA results in the TE mechanical bending of fixed sample, which is recorded. That mechanical deformation is interpreted to be a consequence of the resulting Joule heating, and its amplitude is directly proportional to the applied dc current in the mentioned range. Moreover, the direct proportionality between TE deformation and the resulting increase of temperature was experimentally confirmed. In this way, the new type of temperature microsensor/microactuator working on the principle of TE deformation has been developed. This opens new technological application of microwires as temperature microsensors and temperature-driven microactuators for micro-electro-mechanical system devices

Comparaison de méthodes d'enrichissement des phosphopeptides par IMAC ou TiO2 pour leur analyse par spectrométrie de masse

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Comparative epididymal proteome of monotremes and other mammals including humans

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Human epididymal proteins - secretion, absorption, and luminal fluid composition

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Approches non invasives de l'embryon : protéomique, métabolomique, dialogue ovocyte-cumulus

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