The quantitation of bioavailable metal is difficult with traditional analytical methods. However, the bioavailability of metals is an important factor in the determination of metal toxicity and therefore the detection of bioavailable metals is of interest. The new concept of analyzing bioavailability of heavy metals by creating microbial strains capable of sensing the environment will rely largely upon the functional genomics of a metal resistance genetic system. The greatest advantage is the ability of biosensors to detect the bioavailable fraction of the contaminant, as opposed to the total concentration. Such whole-cell bacterial biosensors will create a clearer picture by providing physiologically relevant data in response to a contaminant.
The essence of all metal resistance genetic systems is the specificity of genetic regulatory elements so that the corresponding metal controls the expression of the uptake or resistance gene products. For example, cobalt-zinc-cadmium resistance operon (czc operon) in Ralstonia sp. CH34 is regulated by a two-component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR. Regulatory genes are arranged in an upstream as well as down-steam regulatory region. Genomics revealed the presence of the czcR and czcS together with czcD constituting the downstream regulatory region. Functional genomics with czcD::lacZ translation fusion and czcS::lux transcriptional fusion enabled the regulation of both genes by heavy metals to be understood .
These systems can be used as the contaminant-sensing component of the biosensor by detecting the substance for which it is designed to detoxify or excrete. The contaminant-sensing component is combined with the reporter genes to create biosensors that can identify toxic substances at very low levels. When the contaminant-sensing component detects the substance, it triggers the reporter gene. In the development of a mercury-specific biosensor, a hypersensitive clone was constructed using the regulatory sequence along with the mercury (Hg+2) uptake genes merTPC of the mercury resistance operon. Such a clone was found responsive to Hg+2 with as low as 0.5 nM — several folds lower than the lowest concentration required to induce the operon without the merTPC.
A reporter gene encodes for a mechanism that produces a detectable cellular response. It determines the sensitivity and detection limits of the biosensor. Specific characteristics are needed for the reporter gene to be used in a biosensor. The gene must have an expression or activity that can be measured using a simple assay and reflects the amount of chemical or physical change. Also, the biosensor must be free of any gene expression or activity similar to the desired gene expression or activity being measured. The suitable reporter genes are (1) lacZ (P-galactosidase); (2) firefly (Photinus pyralis) luciferase, lucFF; and (3) bacterial luciferase, luxAB [342-43].
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