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              *Promoter Characterization
              *Operation Characterization


         Project > Biosensor

Biosensor Introduction

    Sensing techniques form an integrated part of our modern life. We like to be accurately and constantly informed about the quality, security and composition of products that we consume or encounter in our daily life. Medical tests need to provide instantaneous answers on health parameters, blood values or presence of potential pathogenic organisms. Sensors come in thousand and more forms and shapes, principles and output. Future demand calls for further miniaturization, continuous sensing, rapidity, increased sensitivity or flexibility.
    One of the emerging domains in sensing technology is the use of living microbial cells or organisms(van der Meer and Belkin). A biosensor is a measurement device or system that is composed of a biological sensing component, which recognizes a chemical or physical change, coupled to a transducing element that produces a measurable signal in response to the environmental change(Daunert et al., 2000). It is only since the last twenty years that living cell-based sensing assays have gained impetus and developed into a scientific and technological area by itself.
    The question arises here is why one would use living cells and organisms for sensing? What are the specific purposes for basing sensing methods on living cells and what are the advantages that cellular-based sensing can have over other sensing techniques?
    Testing for toxic pollution such as heavy metals is commonly performed with chemical test kits of unsatisfying accuracy (Stocker et al., 2003). Normally, costly equipment is also needed. Instead, bacterial biosensors are easily produced low cost, simple, and highly accurate devices. For example, both laboratory and field studies have demonstrated arsenic detection limits in bacterial bioreporter assays of close to 5 nM, much lower than the drinking water standard of 10 ug, making these assays ideal for analysing large numbers of samples in developing countries facing arsenic contamination of their potable water sources. Some bioreporter assays (for example, for Hg or As) have excellent measurement accuracies and compound detection specificities, and some may even compete with chemical methods. Namely, bacterial sensor-reporters, which consist of living micro-organisms genetically engineered to produce specific output such as GFP fluorescence or colors that can be distinguished by naked eyes, offer an interesting alternative for heavy metal detection.

Fig.1 Genetically engineered bacteria, tailored to respond by a quantifiable and easily recognizable signal to the presence of heavy metal, may serve as powerful tools for heavy metal detection and further assessment of the extent and the implications of environmental pollution.

    Despite numerous proofs of principle, however, most bioreporters have remained confined to the laboratory (van der Meer and Belkin). As assay parameters such as induction time, cell number and cellular activity can not always be controlled in a suffciently rigorous manner; bacterial bioreporter assays on unknown samples are always performed in conjunction with a set of external calibrations. Also bacterial reporters have NOT been rational enough in design and NOT complex enough in function (Chakraborty et al., 2008; Diesel et al., 2009; Sharon Yagur-Kroll, 2010). Additionally, we notice that genetic manipulation in this field needs streamline methods (Hansen and Sorensen, 2000), which means even a limited standardization only for heavy metal biosensor construction is highly desired. It is also notable that it's time for a series of issues on the field application to be taken into consideration, such as optimization of preservation conditions of bioreporter bacteria (Kuppardt et al., 2009).
    During this summer, we engineered our bacteria to resolve those hard truths mentioned above. MerR family transcription factors (TFs) was exploited to construct a series of biosensor for heavy metal detection (Hobman, 2007; Hobman et al., 2005), based on the principle of biological network control (Chikofsky and Cross, 1990).
    The MerR family of proteins is a group of transcriptional factors that control metal ion, radical, and small organic molecule concentrations inside bacterial cells (Nascimento and Chartone-Souza, 2003). The MerR proteins are present in most bacterial genomes and these proteins typically regulate defensive systems against toxic or elevated levels of metal ions. Members from this protein family have been found to sense and control levels of Cd2+, Zn2+, Co2+, Cu+ , Ag+ , Au+ , Hg2+, and Pb2+ ions inside various bacteria(Nascimento and Chartone-Souza, 2003).
    MerR is the archetype of the MerR family of proteins that detoxify mercury(II) ions inside bacteria (Fig 2). The merR gene was first identified in the transposons Tn501 from Pseudomonas aeruginosa and Tn21 from the Shigella flexneri R100 plasmid. In our project, MerR protein comes from the latter. MerR can recognize Hg2+ at 10^8 M concentration even in the presence of mM concentrations of small molecular thiol competing ligands(Nascimento and Chartone-Souza, 2003). The protein binds Hg2+ hundred times more selectively over other metal ions such as Cd2+, Pb2+, Zn2+, Co2+, Ni2+.

Fig 2. Function of the MerR family of proteins. (a) The archetype of the MerR family of transcriptional activators is the regulator of mercury resistance (mer) MerR itself. Addition of Hg2+ to the MerR dimer leads to a conformational change of the protein that enables RNA polymerase (RNAP) to initiate the transcription of the downstream mercury(II) detoxification and efflux genes. (b) The lead resistance operon in R. metallidurans strain CH34 ( pbr) is regulated by PbrR, a protein that mediates Pb2+-inducible transcription from its divergent promoter. (c) Sequence alignment of MerR from transposon Tn21 with PbrR691 from R. metallidurans CH34. The conserved Cys residues engaged in metal binding are highlighted in yellow. Adapted from Peng Chen and Chuan He, 2008.

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