Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma cruzi</it>, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial <it>in silico </it>studies of the <it>T. cruzi </it>genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in <it>T. cruzi </it>are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems.</p> <p>Results</p> <p>While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes.</p> <p>Conclusion</p> <p>Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of <it>T. cruzi</it>. This methodology should greatly facilitate reverse genetic studies in <it>T. cruzi</it>.</p

Testing finite state machines presenting stochastic time and timeouts

In this paper we define a formal framework to test implementations that can be represented by the class of finite state machines introduced in [10]. First, we introduce an appropriate notion of test. Next, we provide an algorithm to derive test suites from specifications such that the constructed test suites are sound and complete with respect to two of the conformance relations introduced in [10]. In fact, the current paper together with [10] constitute a complete formal theory to specify and test the class of systems covered by the before mentioned stochastic finite state machines

A test generation framework for quiescent real-time systems

We present an extension of Tretmans’ theory and algorithm for test generation for input-output transition systems to real-time systems. Our treatment is based on an operational interpretation of the notion of quiescence in the context of real-time behaviour. This gives rise to a family of implementation relations parameterized by observation durations for quiescence. We define a nondeterministic (parameterized) test generation algorithm that generates test cases that are sound with respect to the corresponding implementation relation. Also, the test generation is exhaustive in the sense that for each non-conforming implementation a test case can be generated that detects the non-conformance

Molecular Structure and Vibrational Spectra of Iodotrimethylgermane (GeIMe 3 ) by Theory and Experiment

The geometry of iodotrimethylgermane has been determined by experimental and computational methods. Fourier transform infrared spectra have been recorded over a range of temperatures along with the Raman spectrum to obtain comprehensive vibrational data for the fundamental modes. The stretching, rocking, and deformation bands of the methyl groups have been resolved into their components with the aid of lowtemperature infrared spectroscopy using Fourier self-deconvolution and curve-fitting methods. The optimized geometries and vibrational harmonic frequencies were calculated by density functional theory methods employing Pople-type basis sets, as well as those with descriptions for an effective core potential describing both germanium and iodine atoms. A scaled quantum mechanical analysis was carried out to yield the best set of harmonic force constants and obtain a transferable set of scale factors that can be applied to the (CH3)3- GeX (X ) H, Cl, Br, I) series. GeX (X ) H, Cl, Br, I) series. GeX (X ) H, Cl, Br, I) series. GeX (X ) H, Cl, Br, I) series. GeX (X ) H, Cl, Br, I) series. GeX (X ) H, Cl, Br, I) series. GeX (X ) H, Cl, Br, I) series. 3)3- GeX (X ) H, Cl, Br, I) series.) H, Cl, Br, I) series.Fil: Roldán, María L.. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Física; ArgentinaFil: Brandán, Silvia A.. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Física; ArgentinaFil: Masters, Sarah L.. University of Edinburgh; Reino UnidoFil: Wann, Derek A.. University of Edinburgh; Reino UnidoFil: Robertson, Heather E.. University of Edinburgh; Reino UnidoFil: Rankin, David W. H.. University of Edinburgh; Reino UnidoFil: Ben Altabef, Aida. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Química del Noroeste. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química del Noroeste; Argentin

On conformance testing for timed systems

Contains fulltext : 72665.pdf (publisher's version ) (Closed access)6th International Conference, FORMATS 2008, 15 september 200

Model Based Testing with Labelled Transition Systems

Model based testing is one of the promising technologies to meet the challenges imposed on software testing. In model based testing an implementation under test is tested for compliance with a model that describes the required behaviour of the implementation. This tutorial chapter describes a model based testing theory where models are expressed as labelled transition systems, and compliance is defined with the ‘ioco’ implementation relation. The ioco-testing theory, on the one hand, provides a sound and well-defined foundation for labelled transition system testing, having its roots in the theoretical area of testing equivalences and refusal testing. On the other hand, it has proved to be a practical basis for several model based test generation tools and applications. Definitions, underlying assumptions, an algorithm, properties, and several examples of the ioco-testing theory are discussed, involving specifications, implementations, tests, the ioco implementation relation and some of its variants, a test generation algorithm, and the soundness and exhaustiveness of this algorithm