Spatial expression of a mercury-inducible green fluorescent protein within a nanoporous latex-based biosensor coating.
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.
Schottel
Janet L
JL
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Ave., St Paul, MN 55108, USA. schot002@umn.edu
Orwin
Paul M
PM
Anderson
C Ron
CR
Flickinger
Michael C
MC
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D018914:Cells, Immobilized / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007840:Latex / Q000737:chemistry; D008628:Mercury / Q000378:metabolism*; D018613:Microscopy, Confocal; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; D007840:Latex; D049452:Green Fluorescent Proteins; D008628:Mercury