PCR-based gene synthesis to produce recombinant proteins for crystallization

<p>Abstract</p> <p>Background</p> <p>Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves.</p> <p>Results</p> <p>A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille) domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA). The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to <it>in vivo </it>homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in <it>E. coli </it>and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis.</p> <p>Conclusion</p> <p>We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.</p

Ray tracing 3D source modelling for optical reflectance sensing with wireless ranging application

This study delivers a powerful comparison case for six of the most common ray tracing (RT) source models. It demonstrates that in the early stages of the RT algorithm, when only the ray-geometry intersection and ray-reflectance are introduced, the ray source modelling is a pivotal event in the simulation. The six models are compared in a large three-dimensional (3D) scenario of the well-known double-slit experiment, with the comparison metrics delivered by the number of rays that intersect the back screen and the total simulation time. The numerical results for a variable number of 2, 000; 10, 000; 25, 000 and 100, 000 rays that emulate each of the six source models, are accompanied by the simulation's visual output samples to eliminate abstract ambiguities. This work's main contribution applies directly to the RT simulation for wireless ranging, since scientific programming environments such as MA TLAB are extensively utilised in this research field, which provide the required modelling customisation. Moreover, for machine sensing areas involving optical ranging or light detection and ranging (LIDAR) mapping, the presented study provides valuable information about efficient modelling for ray fascicle launching. Furthermore, since RT simulations enable the latest performances in the gaming and animation industries, the basic and clear information presented in this work supports the next generation of their developers in the delivery of hardware and software implementations

Position discrimination of a 2.4 GHz IEEE 802.15.4 RF mobile source inside-outside a vehicle

Thanks to the recent advancements in the automotive industry, in smart city infrastructure and in electronics miniaturization, low-power wireless sensors are becoming a reference sensing technology connecting the internet of things (IoT) with the conventional world. This study provides an empirical solution to the modern radio location problem of inside-outside position discrimination for a mobile radio frequency (RF) source. The solution is delivered by a detection system that is fully enclosed inside a modern vehicle cabin, whereas the RF ranging is based solely on the received signal strength indicator (RSSI) and the individual sensor’s directivity achieved through shielding. The RF detection system is provided through a low-power wireless sensing network as a complete 2.4 GHz IEEE 802.15.4 solution, anticipating the future integration of this technology in the next generation of smartphones. The RSSI fingerprinting database, which is derived from empirical outdoor measurements for a range up to 5 m, delivers a consistent performance inside the highly RF-reflective vehicle cabin by exploiting the sensor position and directivity, focused on the front of each seat to avoid future human interference. Moreover, a theoretical propagation model based on Friis’ transmission equation constructed on system parameters shows a high correlation with the RSSI fingerprinting experimental model, supporting the consistency of the empirical model, and demonstrating a similar high inside-outside discrimination. The decision algorithm logics used for inside-outside discrimination illustrate a strong example for sensor group decision based on two spatial thresholds: maximum detection range for outside discrimination and the cabin width for inside discrimination. This study’s location system design creates exploitation possibilities beyond the vehicle environment. Various applications that require complete sensor encasement, such as road flushed traffic sensors or underground systems..

Thermococcus Thioreducens sp. Nov., a Novel Hyperthermophilic, Obligately Sulfur-reducing Archaeon from a Deep-sea Hydrothermal Vent

A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P was isolated from black smoker chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 N, 33.9 W). The cells of strain OGL-20P(sup T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within the pH range 5.0-8.5 (optimum pH 7.0), NaCl concentration range 1-5 % (w/v) (optimum 3%), and temperature range 55-94 C (optimum 83-85 C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, iron (III) or nitrate. Proteolysis products (peptone, bacto-tryptone, casamino-acids, and yeast extract) are utilized as substrates during sulfur-reduction. Strain OGL-20P(sup T) is resistant to ampicillin, chloramphenicol, kanamycin, and gentamycin, but sensitive to tetracycline and rifampicin. The G+C content of DNA is 52.9 mol%. The 16S rRNA gene sequence analysis revealed that strain OGL-20P(sup T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(sup T) represents a new separate species within the genus Thermococcus, and propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(sup T) (= ATCC BAA-394(sup T) = JCM 12859(sup T) = DSM 14981(sup T))