Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius

Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli–Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2–0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30–50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L−1 culture. In addition we also determined the promoter strength of several constitutive promoters

“Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics

Fasergekoppeltes Terahertz-Spektroskopiesystem

The use of broadband laser pumped Terahertz (THz) systems for industrial applications strongly relies on the reliability and stability of the system. It has to be compact and easy to be implemented into the particular application. Fiber-coupled THz systems are the key to widespread use of these systems. In order to couple THz emitters and detectors to fibers with lengths of several meters the dispersion and the nonlinear properties of fibers have to be taken into account. This is discussed in this article. As a possible application of the fiber-coupled Terahertz system in the field of nondestructive testing the measurements of the thicknesses of thin films are shown. These measurements are performed in both transmission and reflection

A 3D THz image processing methodology for a fully integrated, semi-automatic and near real-time operational system

The present study proposes a fully integrated, semi-automatic and near real-time mode-operated image processing methodology developed for Frequency-Modulated Continuous-Wave (FMCW) THz images with the center frequencies around: 100 GHz and 300 GHz. The quality control of aeronautics composite multi-layered materials and structures using Non-Destructive Testing is the main focus of this work. Image processing is applied on the 3-D images to extract useful information. The data is processed by extracting areas of interest. The detected areas are subjected to image analysis for more particular investigation managed by a spatial model. Finally, the post-processing stage examines and evaluates the spatial accuracy of the extracted information.© (2012) COPYRIGHT Society of Photo-Optical Instrume ntation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

Towards 3-D THz volume inspection for process control

We report on the development of a stepped frequency continuous wave THz imaging system for process control in industrial environments. The system is based on a sparse line array consisting of 12 emitters and 12 heterodyne receivers operating in a frequency range between 75 GHz and 110 GHz. The applied multiple-input and multiple-output technique allows the reconstruction of THz images with 144 virtual pixels per line, while a band-conveyer below the line array provides the required feed motion of the sample for 3-D data acquisition

Terahertz time domain spectroscopy for nondestructive testing and sensing applications

Terahertz spectroscopy and imaging using innovative photonic THz sources and detectors have turned out to be very useful techniques for applications in nondestructive testing and sensing. Typical examples of THz imaging and spectroscopy are presented

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80°C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon

DEVELOPMENT AND OPTIMIZATION OF THZ NDT ON AERONAUTICS COMPOSITE MULTI-LAYERED STRUCTURES

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