Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the productof the Escherichia coli aroA gene, has been overproduced inE.coli BL21(DE3) under the control of the T7 gene 10 promoterand ribosome binding site, to a level of {small tilde}50% oftotal cell protein. EPSP synthase is the primary target of thepost-emergence herbicide, glyphosate, commonly known as Roundup^TM.A simple two step purification is described, which results in99% pure homogeneous protein (as determined by PAGE). The integrityof the protein has been compared with previously characterizedprotein from .E.coli AB2829(pKD501) by determination of itskinetic parameters, N-terminal protein and DNA sequences, aminoacid analysis and ¹³C-NMR spectroscopy. This new overproducingstrain readily provides the gram quantities of highly pure proteinrequired for NMR studies of the active site and the developmentof novel time-resolved solid-state NMR techniques currentlyunderway in this laboratory.     

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase

Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26–Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257–Pro258and Asp293–Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257–Pro258and Asp293–Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wild–type glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5.     

Ultrafiltration of lake water for potable water production

Ultrafiltration with coagulation or powdered activated carbon pretreatment is used to treat water from a public water supply. While UF without pretreatment is effective in removing turbidity, pretreatment prior to UF greatly enhances the removal of natural organic matter and trihalomethane precursors. Short-term flux loss can be minimized with backflushing, while long-term irreversible fouling seems to be affected most by the type of membrane polymer.     

The importance of axial misalignment on the long term strength of polyethylene pipe butt fusion joints

The importance of axial misalignment at polyethylene pipe butt fusion joints has been assessed by undertaking elevated temperature lifetime tests. Both medium and high density polyethylene pipes were fusion joined to give aligned and controlled misaligned butt joints. These were tested under either a constant or fluctuating internal pressure loading using conditions that induced failure by slow, stable crck growth. It was observed that the lifetime of a butt joined system depends upon both the internal pressure or pressure range applied and the level of misalignment at the butt fusion joint. Increasing either the internal pressure (range) or the misalignment reduced system performance. These two variables of misalignment and internal pressure (range) may be incorporated into a single parameter, the amplified axial stress (or stress range) at the butt joint. This amplified or butt joint axial stress (or stress range) may be derived by considering the additional bending stresses introduced at the butt joint by virtue of misalignment combined with the axial stress loading.     

Experiments of a prototype wind-driven reverse osmosis desalination system with feedback control

A prototype wind-powered reverse osmosis desalination system was constructed and tested on Coconut Island off the northern coast of Oahu, Hawaii, for brackish water desalination. The system has four major subsystems: a multi-vaned windmill/pump, a flow/pressure stabilizer, a reverse osmosis module, and a control mechanism. The feedback control mechanism, developed by this study, allowed this prototype system to be operated satisfactorily under mild ambient wind of 5 m/s or less. No auxiliary power source was needed. The system operational data showed that at an average wind speed of 5 m/s, brackish feedwater at a total dissolved solids concentration of 3000 mg/1 and at a flow rate of 13 1/min could be processed by this system. The average rejection rate of this prototype system was 97% and the average recovery ratio was 20%. The energy efficiency of the system was measured at 3 5%, which is comparable to the typical energy efficiency of well-operated multi-vaned windmills. Generally, the system's energy efficiency decreases as wind speed increases.     

Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

现代生活的绘画——从马奈及其追随者的艺术看巴黎

本文是美国著名艺术理论家、批评家T.J.克拉克同名著作修订版的导论部分,探讨了印象派绘画与巴黎的关系.     

Using a conductive sphere as a probe to characterize the sensitivity of soft piezoresistive films

Flexible piezoresistive films, such as, carbon black/polydimethylsiloxane (C-PDMS) composites, are often used as skin analogs and integrated into complex array sensors for tactile sensing. The uniformity of the sensor characteristics heavily depends on the homogeneity of the composite. Therefore, the ability to locally characterize a film that will be integrated into a complex force sensor could be critical. Here, a method to characterize the local sensitivity of flexible piezoresistive films is presented. Using a conductive sphere, which was chosen over a flat probe to eliminate misalignment issues, the surface of a thin film composite is indented to characterize the change in resistivity in terms of average strain. Experiments were performed with 15 and 18 wt% carbon black C-PDMS films of varying thickness. The contact radius of the probe with the piezoresistive film was estimated using the Johnson-Roberts-Kendall contact theory. Theoretical contact area estimates were found to agree with contact radius measurements carried out using optically transparent PDMS films observed through an optical microscope. Results show that C-PDMS with 15 wt% carbon black exhibit a higher rate if change of resistivity and gauge factor than films of same thickness with 18 wt% carbon black. On the other hand, thicker films exhibit higher gauge factors for the two tested carbon black contents. Tests carried out at multiple locations yielded consistent sensitivity values, making these types of composites suitable for array type force sensors.     

The damping performance of aluminum-based composites

Metal-matrix composites may offer better damping properties than unreinforced alloys. Because damping properties (and metal-matrix composites) are becoming important in airframe design, the damping capabilities of a number of aluminum-matrix composites were measured over a wide range of frequencies at low strain amplitudes, using a new laser vibrometer technique. Silicon carbide and alumina reinforcements resulted in a material with damping properties similar to that of unreinforced aluminum 6061-T6, but unidirectional and planar-random graphite continuous-fiber reinforcements increased the damping by 5 and 14 times, respectively. The increased damping of the continuous fiber composites is attributed to the absence of interfacial reaction resulting from the high-pressure infiltration method used for their manufacture.