Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.     

The Influence of the Amount of Fluoride Catalyst on the Morphological Properties of the Anilinepropylsilica Xerogel Prepared in Basic Medium

Anilinepropylsilica hybrid powder was synthesized by a sol-gel route in basic medium using HF as catalyst. The effect of the amount of HF catalyst on the morphologies and on organic content was studied. The xerogels were characterized using FTIR, SEM and N₂ adsorption-desorption isotherms. The increase in the HF quantity results in an slight increase in organic content and changes in the particle size, surface area and pore structure.     

Microstructural Evolution in Liquid-Phase-Sintered SiC: Part III, Effect of Nitrogen-Gas Sintering Atmosphere

Effects of N₂ sintering atmosphere and the starting SiC powder on the microstructural evolution of liquid-phase-sintered (LPS) SiC were studied. It was found that, for the β-SiC starting powder case, there was complete suppression of the β→α phase transformation, which otherwise goes to completion in Ar atmosphere. It was also found that the microstructures were equiaxed and that the coarsening was severely retarded, which was in contrast with the Ar-atmosphere case. Chemical analyses of the specimens sintered in N₂ atmosphere revealed the presence of significant amounts of nitrogen, which was believed to reside mostly in the intergranular phase. It was argued that the presence of nitrogen in the LPS SiC helped stabilize the β-SiC phase, thereby preventing the β→α phase transformation and the attendant formation of elongated grains. To investigate the coarsening retardation, internal friction measurements were performed on LPS SiC specimens sintered in either Ar or N₂ atmosphere. For specimens sintered in N₂ atmosphere, a remarkable shift of the grain-boundary sliding relaxation peak toward higher temperatures and very high activation energy values were observed, possibly due to the incorporation of nitrogen into the structure of the intergranular liquid phase. The highly refractory and viscous nature of the intergranular phase was deemed responsible for retarding the solution–reprecipitation coarsening in these materials. Parallel experiments with specimens sintered using α-SiC starting powders further reinforce these arguments. Thus, processing of LPS SiC in N₂ atmosphere open the possibility of tailoring their microstructures for room-temperature mechanical properties and for making high-temperature materials that are highly resistant to coarsening and creep.     

Direct Synthesis and Hydration of Calcium Aluminosulfate (Ca4Al6O16S)

Calcium aluminosulfate (Ca₄Al₆O₁₆S or C₄A₃̄) was prepared by direct synthesis from calcium and aluminum nitrates, and aluminum sulfate. CaAl₄O₇(CA₂) formed as an intermediate at 900°C, and C₄A₃̄ was the main phase after calcination at 1100°C. The specific surface areas after calcination at 1100° and 1300°C were ∼2.5 and 1 m²/g, respectively. Hydration was investigated using XRD, DSC, SEM, conduction calorimetry, and solid-state ²⁷Al MAS-NMR spectroscopy. Calorimetry showed that the induction period was longer than that of a sample prepared using conventional solid-state sintering, and this was attributed to the formation of amorphous coatings. Crystalline hydration products, principally calcium monoaluminosulfate hydrate and aluminum hydroxide, appeared subsequently. Although the induction period was very long, complete hydration occurred as early as 3 d in the sample calcined at 1100°C and was 91% complete in the sample calcined at 1300°C.     

Pressurized fluidized-bed hydro retort ing of raw and beneficiated Eastern oil shales

Bench-scale tests were conducted with raw and beneficiated shales in an advanced multi-purpose research reactor. Raw Alabama shale and raw and beneficiated Indiana shales were retorted at 515 °C using hydrogen pressures of 4 and 7 MPa. Shale feed rates were 15 to 34 kg h⁻¹. High oil yields and carbon conversions were achieved in all tests. Oil yield from Alabama shale hydroretorted at 7 MPa was 200% of Fischer assay. Raw and beneficiated Indiana shales hydroretorted at 7 MPa produced oil yields of 170 and 195% of Fischer assay respectively. Total carbon conversions were >70% for all tests conducted at 7 MPa.     

Upgrading a rutile concentrate produced from Athabasca oil sands tailings

A rutile concentrate recently produced by Lakefield Research Ltd and Syncrude Canada Ltd from athabasca oil sands tailings was characterized and attempts were made to upgrade it to market-grade. The rutile concentrate contained 75.5% TiO₂, 18.7% Fe₂O₃, 1.03% Al₂O₃, 1.94% SiO₂, and 563 ppm Th+U. The amount of rutile (anatase) in the concentrate was estimated to be about 10-17%, and the remaining TiO₂ came from ilmenite in various altered forms. Magnetic separation could remove the Fe from the concentrate but it increased the SiO₂ content from 1.94 to 6.01%. A reverse flotation process was developed to remove the SiO₂. Although the fine dissemination of SiO₂ in the rutile matrix limited the removal of SiO₂, the upgraded rutile concentrate, containing 87-89% TiO₂, could be used as a feedstock to the chloride titanium pigment processes.     

Chemical approaches to triggerable lipid vesicles for drug and gene delivery

Effective drug delivery requires the precise spatial and temporal delivery of therapeutic agents to the target site. To this end, a variety of chemical and physicochemical approaches have been devised to create lipid vesicles (liposomes) that can be triggered to release their contents in a controlled fashion. The triggers include changes in pH, redox potential, temperature, or the level of specific enzymes. We review the chemistries that have recently been applied to exploit the pH and redox potential triggers so as to release vesicle contents in the appropriate biological location.     

Microstress in Reaction‐Bonded SiC from Crystallization Expansion of Silicon

Microstress in reaction‐bonded silicon carbide (RBSiC) has been measured using piezo‐Raman spectroscopy. Compressive microstresses as high as 2 GPa exist in the silicon phase and tensile microstresses as high as 2.3 GPa exist in the SiC phase of RBSiC. This is much larger than expected for thermoelastic microstress from coefficient of thermal expansion mismatch would provide. Instead the microstresses arise from the crystallization of liquid silicon. During the reaction bonding process, not all of the silicon reacts to form SiC and there is liquid free silicon. The phase transformation of the free silicon from liquid to solid has a large volume expansion, which results in large residual microstress within the silicon and SiC phases of RBSiC.     

Effect of clay type on dispersion and barrier properties of hydrophobically modified poly(vinyl alcohol)–bentonite nanocomposites

The oxygen and water vapor permeability at high relative humidity was studied for composite films formed by incorporation of three different bentonites (MMT) into an ethylene‐modified, water‐soluble poly(vinyl alcohol), EPVOH. The oxygen permeability decreased linearly with an increased addition of hydrophilic MMTs. X‐ray diffraction and Fourier transform infrared spectroscopy suggested a homogeneous distribution in the thickness direction with disordered and probably exfoliated structures for hydrophilic MMTs. In contrast, organophilic modified clay showed an intercalated structure with the clay preferentially located at the lower film surface, a combination which was however efficient in reducing the water vapor‐ and oxygen permeabilities at low addition levels. Composite films of EPVOH and Na⁺‐exchanged MMT resulted in high resistance to dissolution in water, which was ascribed to strong interactions between the components resulting from matching polarities. Annealing the films at 120°C resulted in enhanced resistance to water dissolution and a further reduction in oxygen permeability. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42229.     

Microstructural Evolution in Liquid-Phase-Sintered SiC: Part I, Effect of Starting Powder

The effect of starting SiC powder (β-SiC or α-SiC), with simultaneous additions of Al₂O₃ and Y₂O₃, on the microstructural evolution of liquid-phase-sintered (LPS) SiC has been studied. When using α-SiC starting powder, the resulting microstructures contain hexagonal platelike α-SiC grains with an average aspect ratio of 1.4. This anisotropic coarsening is consistent with interface energy anisotropy in α-SiC. When using β-SiC starting powder, the β→α phase transformation induces additional anisotropy in the coarsening of platelike SiC grains. A strong correlation between the extent of β→α phase transformation, as determined using quantitative XRD analysis, and the average grain aspect ratio is observed, with the maximum average aspect ratio reaching 3.8. Based on these observations and additional SEM and TEM characterizations of the microstructures, a model for the growth of these high-aspect-ratio SiC grains is proposed.