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Engineering the effector specificity of regulatory proteins for the in vitro detection of biomarkers and pesticide residues

Chen, Wei, Zhang, Xuanxuan, Xiong, Dandan, Jin, Jian-Ming, Tang, Shuang-Yan
Applied microbiology and biotechnology 2019 v.103 no.7 pp. 3205-3213
DNA, Pseudomonas putida, biomarkers, biosensors, carbaryl, engineering, enzymes, mutants, pesticide residues, regulatory proteins, transcription (genetics), uric acid
Transcriptional regulatory proteins (TRPs)-based whole-cell biosensors are promising owing to their specificity and sensitivity, but their applications are currently limited. Herein, TRPs were adapted for the extracellular detection of a disease biomarker, uric acid, and a typical pesticide residue, carbaryl. A mutant regulatory protein that specifically recognizes carbaryl as its non-natural effector and activates transcription upon carbaryl binding was developed by engineering the regulatory protein TtgR from Pseudomonas putida. The TtgR mutant responsive to carbaryl and a regulatory protein responsive to uric acid were used for in vitro detection, based on their allosteric binding of operator DNA and inducer molecules. Based on the quantitative polymerase chain reactions (qPCRs) output, the minimum detectable concentration was between 1 nM–1 μM and 1–10 nM for uric acid and carbaryl, respectively. Our results demonstrated that engineering the effector specificity of regulatory proteins is a potential technique for generating molecular recognition elements for not only in vivo but also in vitro applications.