PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.     

Improved particle concentration by cascade AC electroosmotic flow

The importance of electrokinetics in microfluidic technology has been growing owing to its versatility and simplicity in fabrication, implementation, and handling. Alternating-current electroosmosis (ACEO), which is the motion of fluid due to the ion movement by an interaction between AC electric field and an electrical double layer on the electrode surface, has a potential for a particle concentration method to detecti rare samples flowing in a microchannel. This study investigates an improved ACEO-based particle concentration by cascade electrokinetic approach. Flow field induced by ACEO and accumulation behavior of particles were parametrically measured to discuss the concentrating mechanism. The accumulation of particles by ACEO can be explained by a balance between the attenuating electroosmotic flow to transport particles and the inherent diffusive motion of the particles, which is hindered due to the near-wall location. Although a parallel double-gap electrode geometry enables particles to be collected at the center of electrode very sharply, it has scattering zones with accumulated particles at sidewalls of the channel. This drawback can be overcome by applying sheath flow or introducing cascade electrode pattern upstream of the focusing zone. As a result, total concentration efficiency was 98.4 % for all the particles flowing in the cascade device. The resultant concentrated particles exist on the electrode surface within 5 μm, and three-dimensional concentration of particle with the concentration factor as large as 700 is possible using a monolithic channel, co-planar electrode, and sheathless solution feeding. This cascade electrokinetic method provides a new and effective preconcentrator for ultra-sensitive detection of rare samples.     

Deterioration and recovery in the resistivity of Al-doped ZnO films prepared by the plasma sputtering

A hot-cathode plasma sputtering technique was used for fabricating the highly transparent and conducting aluminum-doped zinc oxide (AZO) films on glass substrates from a disk-shaped AZO (Al₂O₃: 2 wt.%) target. Under particular conditions where the target voltage was V_T = −200 V and the plasma excitation pressure was P_S = 1.5 × 10⁻³ Torr, the lowest resistivity of 4.2 × 10⁻⁴ Ω cm was obtained at 400 nm, and this was associated with a carrier density of 8.7 × 10²⁰ cm⁻³ and a Hall mobility of 17 cm²/V s. From the annealing experiment of the AZO films in the oxygen and nitrogen gases of the atmospheric pressure it was revealed that both the oxygen vacancies and the grain boundaries in the polycrystalline AZO film played an important role in the electrical properties of the film.     

High-temperature ultra-strength of dual-phase Re0.5MoNbW(TaC)0.5 high-entropy alloy matrix composite

The dual-phase Re0.5MoNbW(TaC)0.5 composite,consisting of refractory body-centered cubic(BCC)high-entropy alloy and carbide with many fine eutectic structures,was successfully synthesized by arc melting.The phase stability,high-temperature mechanical properties and strengthening mechanism of the as-cast composite were studied.The microstructure of the composite remained stable after annealing at 1300℃for 168h.It exhibited remarkably high-temperature strength,yield strength～901 MPa,and true ultimate compressive strength～1186 MPa at 1200℃.The BCC phase and carbide exhibited a semi-coherent interface with good bonding after severe deformation at 1200℃.The dipolar dislocation walls in BCC phase,restricted dynamic interaction between defects in carbide,and the pinning effect of semi-coherent interface offered effective strengthening effects.     

Unified surface anchoring strength from the splay elastic deformation in nematic slabs

Film thickness dependences of the unified surface anchoring strengths from the splay deformation for nematic cells were discussed. Thin nematic parallel (d = 2-50 μm) and wedge (d = 0.3-5 μm) cells with the same solid surface were prepared to evaluate the surface anchoring strength. The surface anchoring strengths from the splay deformations for two kinds of liquid crystals were measured by using the saturation method. The nematogens used were 4-pentyl-4′-cyanobiphenyl and mixture compound ZLI-4792 (Merck Japan Co., Ltd.) with positive dielectric anisotropy. Film thickness dependence of the surface anchoring strength was elucidated on the basis of the conventional ionic surface polarization. It was also discussed that a surface anchoring strength from the splay deformation is to be an intrinsic value defined at an interfacial region and not a value changing with the film thickness.     

Microstructure,mechanical properties,and cytotoxicity of low Young’s modulus Ti–Nb–Fe–Sn alloys

Journal of Materials Science - Ti–26Nb–2Fe–(0, 2, 4, 6, 8)Sn alloys were prepared by arc melting and subjected to homogenization, cold rolling, and solution treatment. The β...     

A method for determining the stress intensity threshold level for fatigue crack propagation

The influence of a decreasing rate of stress intensity factor with crack propagation, $\text{d}\text{K/}\text{d}\text{a}$, on a stress intensity threshold level, $\text{δK}{}_{\mathrm{th}}$, below which fatigue crack propagation becomes insignificant is investigated. Specimens, 200 mm wide, 10 mm thick with a 40 mm-long central crack, are fatigued at the decreasing rates, $\text{∥}\text{d(δK)}\text{d}\text{a∥}$, of 2,44, 5 and 10 kg/mm^5/2 with a peak load control system and a pair of crack followers. In this range of $\text{d}\text{(δK)/}\text{d}\text{a}$, the stress intensity threshold levels, $\text{δK}{}_{\mathrm{th}}$, have the same value regardless of $\text{d}\text{K/}\text{d}\text{a}$. Therefore, the present method of decreasing the stress intensity factor at a constant rate is suitable for determining the characteristic $\text{δK}{}_{\mathrm{th}}$ of materials. Furthermore, the influence of stress ratio, $\text{R}$, is investigated at the decreasing rate, $\text{∥}\text{d}\text{(δK)/}\text{d}\text{a]}$, of 10 kg/mm^5/2.     

Block-pivoted condensation on impedance matrices

Given an n-port of nullity q, the Z -matrix of which is to be sought, we first construct a primitive Z -matrix of order q, and then apply to it the pivotal condensations with respect to q–n unspecified ports, one at a time. The present note discusses the problem of decomposing a network into blocks and then of seeking a port structure so that any pair of unspecified ports taken from different blocks have no coupling in the primitive Z -matrix. This enables us to apply a block-pivoted condensation to the primitive Z -matrix.