Sridhar Ramanathan

Bacterial biosensors for monitoring toxic metals

Biosensors utilize biological components to provide selectivity for monitoring compounds of environmental, clinical and industrial importance. A number of biosensors based on bacteria have recently been developed for monitoring toxic metals in the environment. The advantages and disadvantages of these types of biosensors are discussed.

Peer Reviewed: Applications of Reporter Genes

Reporter proteins can be used in bioanalytical methods to produce signals indicating the presence of a target analyte.

Bioluminescence competitive binding assays for biotin based on photoprotein aequorin

Publisher Summary This chapter describes competitive binding assays for biotin that use the bioluminescent protein aequorin as the label. Heterogeneous and homogeneous competitive binding assays using aequorin bioluminescence have been developed for detection of biotin. In the heterogeneous system, an aequorin–biotin conjugate competes with unlabeled biotin for binding to immobilized avidin, followed by a separation step to remove the unbound conjugate. Bioluminescence assays are best performed on luminometers operated in a photon-counting mode. A variety of such instruments is commercially available. In addition, modern scintillation counters may be used to perform bioluminescence assays. The relative amounts of avidin and biotinylated aequorin (conjugate) influence the response characteristics of the assay. Therefore, the amounts of avidin and biotinylated aequorin must be optimized in order to enhance assay performance. The development of a homogeneous assay for biotin using aequorin is based on the ability of avidin in solution to inhibit the bioluminescence of biotinylated aequorin. To establish the optimal amount of avidin to use in the assay, a binder dilution curve is constructed by varying the amount of avidin in the presence of a fixed amount of biotinylated aequorin, and measuring the degree of signal inhibition.

Recombinant methods in protein and whole-cell biosensing

In this paper, we investigate the use of fluorescently- labeled binding proteins and genetically engineered bacterial cells for sensing of phosphate, glucose, and L- arabinose. To optimize the performance of the labeled binding proteins for biosensing purposes, a few key considerations were taken into account. A site-selective labeling protocol of the fluorescent reporter to the protein was used to ensure that the probe reported from a specific domain of the protein. The labeling sites chosen were hypothesized to undergo a physicochemical change when the biorecognition element binds the analyte. Cysteine mutations were introduced into the binding proteins by site-directed mutagenesis using the polymerase chain reaction. The residues selected were all in close proximity to the binding cleft, a region that is affected the most by the conformational change that accompanies ligand binding. The cysteine residues were then labeled with environment- sensitive fluorophores and changes in the fluorescence properties of the conjugates were monitored and related to the amount of ligand present. The application of microorganisms in sensing systems represent new advances in the development of novel analytical techniques for the detection of a target analyte. In these systems, a genetically engineered organism generates an analytically useful signal when it encounters a specific target substance due to selective recognition and binding properties towards that particular compound. This concept has been demonstrated using an optical bacteria-based sensing system capable of detecting the monosaccharide L-arabinose that employed the green fluorescent protein as a reporter protein.