In Vitro detection of Toxins using Fusion Protein Biosensors

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Oliver Medvedik, Anne Morill, Peter Yeadon

GenspaceNYC, United States

The detection of trace amounts of toxic metalloids such as arsenic, down to less than 10 parts per billion (ppb), can be readily achieved using genetically modified microorganisms. Using strains of E. coli, genetic pathways based on the arsenic binding ArsR repressor have been modified to enable the microorganism to function as a whole cell biosensor, capable of expressing reporter genes in response to environmental toxins. However, some key drawbacks to using such modified organisms in field conditions lie in maintaining strain viability during storage and transport, along with long incubation times (many hours). Furthermore, the necessity of widespread global adaptation of such GMOs to adequately assess contaminated sources of drinking water poses large geopolitical hurdles in many regions.
Our approach has been to design fusion proteins capable of altered binding to immobilized “bait” DNA molecules in vitro in the presence of specific toxins, such as arsenic. Detection that is based directly on protein-DNA interactions would also allow for more rapid signaling of toxins since the system is independent of gene expression. Fusion proteins have been designed that comprise of an enzyme coupled to the arsenic binding repressor ArsR. In the absence of arsenic, ArsR binds tightly to operator sites of the native Ars operon. The presence or absence of an enzyme-DNA complex can then be readily detected using a variety of substrates, based on the particular enzyme chosen, such as glutathione-S transferase. The output of the reaction can be visualized either qualitatively, as a color change, or measured more quantitatively. Using a DNA binding assay based approach to biosensors lends itself readily to further expansion to a wide variety of other DNA binding proteins whose affinity is affected by small molecule binding, such as the MerR repressor and mercury.