Optimizing the reactivity of cell coatings developed as biosensors or biocatalysts requires measurements of gene expression in the immobilized cells. To quantify and localize gene expression within a latex-based mercury biosensor, a plasmid, pmerGFP, was constructed, which contains the green fluorescent protein (GFP) gene under transcriptional control of the mercury resistance operon regulatory sequences. When cells containing this plasmid were exposed to mercuric chloride, GFP synthesis was induced and could be quantified by fluorescence. E. coli strain JM109 (pmerGFP) was mixed with SF091 latex (Rohm & Haas), Tween 20, and glycerol, and coated as an approximate 20-microm thick nanoporous adhesive coating on a polyester substrate. The cell coat was overlaid with a nanoporous topcoat of latex, Tween 20, and glycerol. Different fluorescent microspheres were used to mark the topcoat and cell coat layers of the coating. Upon exposure to mercury(II), cells within the coating were induced to synthesize GFP, and laser scanning confocal microscopy was used to quantify expression spatially within the cell coat. GFP expression in the coatings increased with increasing mercury concentration (2-20 microM), temperature (21-37 degrees C), and time of incubation (0-39 h). There was a gradient of GFP expression through the cell coat with expression higher near the topcoat-cell coat interface relative to the bottom of the cell coat. The topcoat thickness did not significantly affect GFP expression indicating that diffusion of mercury(II) and oxygen through the topcoat was not limiting.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of industrial microbiology & biotechnology|
|State||Published - Apr 2008|
Bibliographical noteFunding Information:
This project was supported by the BioTechnology Institute at the University of Minnesota. We thank Teresa De la Mora-Rey for her help with plasmid construction. We also thank Dr. Mark Sanders (University of Minnesota-College of Biological Sciences, Imaging Center, http://www.cbs.umn.edu/ic/ ) for his advice concerning LSCM.