This work aims to develop a novel double-stranded (ds) DNA detection biosensor using zinc finger proteins (ZFPs) and a full β-lactamase to detect specific DNA sequences that code for antibiotic resistance genes. This project employs three pairs of ZFPs. The three different target locations within the tetracycline (tetM) gene are recognized by the three sets of ZFP pairs. A six finger ZFP binds to 18 base pairs with high specificity, and this project used a pair of two six finger proteins (ZFP(A) and ZFP(B)), which provide the ability to detect 36 continuous base pairs of DNA. A full-length β-lactamase enzyme was linked to the N-terminal of the ZFP(B) detection probe as a signal transducer. The capture probe ZFP(A) was immobilized on an acrylamide gel surface, and ds DNA containing the target sites are applied to the capture probe for binding. After incubation and washing, the detection probe was applied to the bound complex of the capture probe and target DNA. After the surface was washed, the signal produced by a β-lactamase linked to the detection probe was measured in nitrocefin assay, which indicates the binding of target DNA to ZFPs. The array is optimized with varying concentrations of each component, determining the sensitivity and specificity of our system. Another goal of this project is to characterize the full β-lactamase system to evaluate how it compares to the existing method of the SEERLAC system, reassembly of two split enzyme domains. Overall, the broad context and application of this project is to develop an efficient and accessible detection device to indicate the presence of antibiotic resistant bacteria. This has many meaningful applications to be able to quickly and accurately detect antibiotic resistance genes with a quantifiable signal.

Developing a double-stranded DNA biosensor using engineered zinc finger proteins linked to a β-lactamase for detecting antibiotic resistance genes

2019 Spring National Meeting

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