Plain silver surface plasmon resonance for microarray application.

The application scope of surface plasmon resonance (SPR) and SPR imaging (SPRi) is rapidly growing, and tools such as high-performance and low-cost slides could enable more rapid growth of the field. We describe herein a novel silver slide, addressing the inherent instability of plain silver structure by improving adhesion between the glass substrate and the silver layer with a thin buffer layer of gold. Covered by a self-assembled monolayer (SAM) only, SPR characteristics of the slide remain steady for more than 3 months under regular storage. In a bioassay, the slide substantiates the predicted nearly 100% sensitivity improvement over gold slides and exhibits exceptional performance stability as determined by sensitivity and resolution measurements during the extended 40,000 s multicycle experiment. We demonstrate the suitability of this new slide for large-area SPRi, describing analysis results from a 1 296-ligand protein microarray. We predict this slide structure will provide a stable, high-sensitivity solution for high-throughput SPRi applications and other surface analysis platforms.

Isolation of antibiotics turbomycin a and B from a metagenomic library of soil microbial DNA.

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.