Engineering the effector specificity of regulatory proteins for the in vitro detection of biomarkers and pesticide residues
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.
KeywordsTranscriptional regulatory proteins In vitro detection Carbaryl Uric acid qPCR Allosteric binding
We thank Dr. Junying Jia and Dr. Shuang Sun from Institute of Biophysics, Chinese Academy of Sciences, for FACS sorting. We also thank Dr. Guoxia Liu from Institute of Microbiology, Chinese Academy of Sciences for her technical help in the study.
This work was supported by the National Natural Science Foundation of China (Grant Nos. 31501037, 31870072, 21472234, and 21506245).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
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