We present a methodology for quantitative sensing of the contents of a target material (TM) in a given sample which employs biosensing bioluminescent bacteria. These bacteria are genetically engineered to respond to the presence of a specific TM in their microenvironment by producing bioluminescence. Herein, we extend this methodology to include quantitative sensing of the TM content in the inspected sample by exploiting the dependence of the bioluminescence produced by the bacteria on the content of the TM in the inspected sample. However, employing bacteria as precise measurement devices is inherently problematic, as the signal they produce varies between different batches of bacteria, and changes as the batch ages. Moreover, As the methodology is designed for outdoor operation, the sensitivity of the bacteria response to changes in the environmental conditions needs to be taken into account. These hurdles are overcome in a special optoelectronic sensor which measures in parallel the responses produced by the inspected sample, and a standard sample containing a known quantity of the TM. Both measurements are conducted by identical sensing channels using bacteria from the same batch, and under the same environmental conditions. The “standard ratio” (SR) defined as the ratio between the maximum responses of the inspected sample and the standard sample was found to be independent of the batch and environmental conditions. A calibration curve of the SR vs. the TM concentration in a set of preprepared samples is used to gauge SR at the sensor output to the TM concentration in the inspected sample.

We describe a biosensing module in which live bacteria, genetically "tailored" to respond to the presence of a specific target material, constitute the core sensing element, reporting their response by bioluminescence. The module is constructed of two channels: an 'induced' channel that measures the bioluminescent light emitted by bacteria exposed to the inspected area, and a 'reference' channel that measures in parallel the bioluminescent light emitted spontaneously by bacteria of the same batch. This enables to overcome signal variations generated by different batches of bacteria, and due to varying environmental operating conditions. A special low-noise optoelectronic circuit was constructed to detect the bioluminescence emitted by the bacteria in both channels. The bacteria are encapsulated in polymer beads that also contain nutrients and water, enabling long-term maintenance-free operation. The beads are packaged in special cassettes at the bottom of the module, so that the induced channel cassette is in direct contact with the ground underneath the module, whereas the reference channel cassette is isolated from the ground. The module contains, in addition, a digital signal processing unit, and a wireless communication unit. The module is designed to operate outdoors as an autonomous network element designed for large scale in-situ deployment. The module described herein was developed for the detection of buried landmines, by sensing the presence of 2,4-dinitrotoluene (DNT) vapors released by the mine, accumulating in the ground above it. Detection of DNT in the sub-ppm range is demonstrated.

Cellulose-binding domains have been isolated from various cellulases, and proteins, which lack hydrolytic activity. The hypothesis that a cellulose-binding domain can be used to alter surface and mechanical properties of paper was tested. Two cellulose-binding domains from Clostriium cellulovorans were fused to form a cellulose crosslinking protein (CCP). The recombinant bifunctional cellulose-binding protein was expressed in E. coli, appliedby immersion onto Whatman cellulose filter paper, and its mechanical properties were tested. The purified protein improved the treated paper's mechanical properties (tensile strength, brittleness, Young's modulus and energy to break). In addition, cellulose crosslinking protein treatment was shown to transform filter paper into a more water-repellent paper. The binding of cellulose-binding domains to cellulose under a wide range of envi-ronmentalconditions, without the need for chemical reactions, and its biodegradability make them attractive moieties for the design of a new class of paper-modification materials.p>

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot.

s-Triazines including atrazine are heavily used agricultural herbicides, and their extensive removal from industrial wastewater is required before these effluents can be disposed. The use of enzymes for this purpose is an important potential alternative to conventional methods of detoxification. The purpose of the present study was to develop an enzyme-based technology for the treatment of atrazine contaminated water. In this paper we describe the construction and expression of two fusion proteins which dechlorinate atrazine while being firmly bound to an insoluble cellulose matrix. Dechlorination of atrazine produces in one enzymatic step the nonregulated compound hydroxyatrazine from the regulated mother compound, atrazine.

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.

The heat shock protein hsp60 plays a functional role in insulin-dependent diabetes mellitus. The hsp60 epitope p277 (aa 437–aa 460) is effective in vaccinating mice against diabetes. A synthetic peptide gene (p277) that encodes the human hsp60 epitope was cloned to the 3′ end of the cellulose-binding domain gene (cbd). CBD-p277 was overexpressed inEscherichia coliand purified on a cellulose column. A methionine at the C-terminal end of CBD enabled CNBr cleavage between CBD and p277. After CNBr cleavage, free CBD and residual uncleaved CBD-p277 were recovered by cellulose chromatography. The p277 peptide was further purified on a RPC–FPLC column. The molecular weight of the recombinant peptide was confirmed by electrospray mass spectrometry. The recombinant peptide was found to be biologically active in assays involving clone C9 T-cell proliferation, lymph-node cell proliferation, and antibody production. Thus the use of CBD as an affinity tag and the utilization of affordable cellulose matrices offers an attractive method for the production and purification of recombinant peptides.

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 &mgr;g mL-1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 &mgr;g mL-1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.

Recombinant cellulose-binding domain (CBD) was found to modulate the elongation of different plant cells in vitro. In peach pollen tubes, maximum elongation was observed at 50 mu g/ml CBD. Pollen tube-staining with calcofluor indicated loss of crystallinity in the tip zone of CBD-treated pollen tubes. At high concentrations (100 - 500 mu g/ml), CBD dramatically inhibited root elongation in a dose-responsive manner. However, between 1x10(2)-100 mu g/ml CBD had the opposite effect on root hair elongation as compared to its effect on root elongation. CBD competes with xyloglucan (XG) for binding to cellulose. When CBD was added first to the cellulose, increasing concentrations of CBD resulted in increasing amounts of unbound XG. However, when XG was added first, increasing concentrations of CBD did not affect the level of unbound XG. The level of unbound CBD was higher when XS was added first to the cellulose.