Secreted beta-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment.

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express beta-galactosidase (EC 3.2.1.23) at temperatures below 25 degrees C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a beta-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding beta-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of beta-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42 degrees C, but with short-term stability at temperatures above 25 degrees C.

Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems.

BACKGROUND: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface. RESULTS: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated. CONCLUSION: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.