Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

Membrane osmometry is used to measure osmotic pressures of dilute solutions containing quasi-spherical CdSe nanocrystals covered with polymer brushes in toluene in the range 31 to 45°C. Osmotic-pressure data, as a function of nanocrystal concentration, yield the molecular weight and the osmotic second virial coefficient of the nanocrystals; the latter is related to the potential of mean force between two nanocrystal particles in dilute solution. Coupled with molecular-weight data, extinction coefficients and oscillator strengths are also obtained for nanocrystals of various sizes in toluene. CdSe nanocrystal sizes were obtained either from Transmission Electron Microscopy or from correlations between the wavelength of the absorbing peak and nanocrystal size. Osmotic-pressure data are reduced with a simple perturbed hard-sphere equation of state; the perturbation is due to long-range (London dispersion) attraction and a shortrange interaction potential. The only adjustable parameter, the strength of this short-range potential, shows two-body repulsion or attraction, depending on the sample and on solution conditions.

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The biochemical and morphological differentiation of actinomycetes makes them academically and economically interesting. Their secondary metabolites provide the majority of medically and agriculturally important antibiotics (streptomycete genes may also be the primary source of clinically important antibiotic resistance); their complex morphological developmental cycle involves a series of changes from vegetative mycelial growth to spore formation. Recombinant DNA technology would add a powerful new dimension to the analysis of these various aspects of actinomycete biology and would also facilitate the development of industrial strains with increased antibiotic yield, or capable of making new antibiotics. For most of these purposes, cloning of genes within and between actinomycetes is required to study the expression of particular genes in genetic backgrounds defined by mutations of the characters under study. To achieve this, we have now developed a method for molecular cloning involving the transfer of genes between unrelated streptomycetes.

Transcriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6-dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co-ordinate regulation of genes for erythromycin production and resistance. ermEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site-directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome-binding site to give rise to the full-length 23S rRNA methylase. Deletion of the -35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the 'extended -10' class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their -10 regions and which initiate transcription seven nucleotides downstream of the -10 region.

This paper evaluates the energy consumption and battery performance of city transit electric buses operating on real day-today routes and standardized bus drive cycles, based on a developed framework tool that links bus electrification feasibility with real-world vehicle performance, city transit bus service reliability, battery sizing and charging infrastructure. The impacts of battery capacity combined with regular and ultrafast charging over different routes have been analyzed in terms of the ability to maintain city transit bus service reliability like conventional buses. The results show that ultrafast charging via frequent short-time boost charging events, for example at a designated bus stop after completing each circuit of an assigned route, can play a significant role in reducing the battery size and can eliminate the need for longer duration charging events that would cause schedule delays. The analysis presented shows that significant benefits can be realized by employing multiple battery configurations and flexible battery swapping practices in electric buses. These flexible design and use options will allow electric buses to service routes of varying city driving patterns and can therefore enable meaningful reductions to the cost of the vehicle and battery while ensuring service that is as reliable as conventional buses.

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