Applied Microbiology and Biotechnology

, Volume 103, Issue 7, pp 3205–3213 | Cite as

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

  • Wei Chen
  • Xuanxuan Zhang
  • Dandan Xiong
  • Jian-Ming JinEmail author
  • Shuang-Yan TangEmail author
Methods and protocols


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.


Transcriptional 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.

Funding information

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.

Supplementary material

253_2019_9679_MOESM1_ESM.pdf (527 kb)
ESM 1 (PDF 526 kb)


  1. Banasik M, Sachadyn P (2016) A colorimetric microplate assay for DNA-binding activity of His-tagged MutS protein. Mol Biotechnol 58:521–527CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  3. Casarett LJ, Klaassen CD, Amdur MO, Doull J (1986) Casarett and Doull's toxicology: the basic science of poisons. Collier Macmillan, TorontoGoogle Scholar
  4. Chen D, Wei XB, Zou J, Wang R, Liu X, Xu XF, Lu JJ, Wang ZH, Tang BS, Wang B, Jin KL, Wang Q (2015a) Contra-directional expression of serum homocysteine and uric acid as important biomarkers of multiple system atrophy severity: a cross-sectional study. Front Cell Neurosci 9:247CrossRefGoogle Scholar
  5. Chen W, Zhang S, Jiang PX, Yao J, He YZ, Chen LC, Gui XW, Dong ZY, Tang SY (2015b) Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis. Metab Eng 30:149–155CrossRefGoogle Scholar
  6. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645CrossRefGoogle Scholar
  7. Dietrich JA, McKee AE, Keasling JD (2010) High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu Rev Biochem 79:563–590CrossRefGoogle Scholar
  8. Erden PE, Kilic E (2013) A review of enzymatic uric acid biosensors based on amperometric detection. Talanta 107:312–323CrossRefGoogle Scholar
  9. Frandoloso R, Martinez-Martinez S, Rodriguez-Ferri EF, Yubero S, Rodriguez-Lazaro D, Hernandez M, Gutierrez-Martin CB (2013) Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin prevent the expression of proinflammatory chemokines and cytokines in pigs. Clin Dev Immunol 2013:132432CrossRefGoogle Scholar
  10. Gautam K, Shetty D, Trivedi VD, Varunjikar M, Phale PS (2017) Compartmentalization of carbaryl degradation pathway: molecular characterization of inducible periplasmic carbaryl hydrolase from Pseudomonas spp. Appl Environ Microbiol 84:e02115–e02117Google Scholar
  11. Guo XS, Zhang XY, Cai Q, Shen T, Zhu SM (2013) Developing a novel sensitive visual screening card for rapid detection of pesticide residues in food. Food Control 30:15–23CrossRefGoogle Scholar
  12. Hoa XD, Kirk AG, Tabrizian M (2007) Towards integrated and sensitive surface plasmon resonance biosensors: a review of recent progress. Biosens Bioelectron 23:151–160CrossRefGoogle Scholar
  13. Holubova-Mickova B, Blazkova M, Fukal L, Rauch P (2010) Development of colloidal carbon-based immunochromatographic strip for rapid detection of carbaryl in fruit juices. Eur Food Res Technol 231:467–473CrossRefGoogle Scholar
  14. Hu CY, Chang YJ, Yin LT, Tsao CY, Chang CH (2005) Optimal design of nickel-coated protein chips using Taguchi approach. Sensors Actuators B Chem 108:665–670CrossRefGoogle Scholar
  15. Khatun MA, Hoque MA, Zhang Y, Lu T, Cui L, Zhou NY, Feng Y (2018) Bacterial consortium-based sensing system for detecting organophosphorus pesticides. Anal Chem 90:10577–10584CrossRefGoogle Scholar
  16. Li H, Liang CN, Chen W, Jin JM, Tang SY, Tao Y (2017a) Monitoring in vivo metabolic flux with a designed whole-cell metabolite biosensor of shikimic acid. Biosens Bioelectron 98:457–465CrossRefGoogle Scholar
  17. Li SS, Zhou L, Yao YP, Fan KQ, Li ZL, Zhang LX, Wang WS, Yang KQ (2017b) A platform for the development of novel biosensors by configuring allosteric transcription factor recognition with amplified luminescent proximity homogeneous assays. Chem Commun 53:99–102CrossRefGoogle Scholar
  18. Liang C, Xiong D, Zhang Y, Mu S, Tang SY (2015) Development of a novel uric-acid-responsive regulatory system in Escherichia coli. Appl Microbiol Biotechnol 99:2267–2275CrossRefGoogle Scholar
  19. Liu N, Gao ZX, Ma HW, Su P, Ma XH, Li XL, Ou GR (2013) Simultaneous and rapid detection of multiple pesticide and veterinary drug residues by suspension array technology. Biosens Bioelectron 41:710–716CrossRefGoogle Scholar
  20. Mahr R, Frunzke J (2016) Transcription factor-based biosensors in biotechnology: current state and future prospects. Appl Microbiol Biotechnol 100:79–90CrossRefGoogle Scholar
  21. Mousavizadeh K, Dehpour AR, Minagar A, Ghafourifar P (2003) Uric acid: a novel treatment strategy for multiple sclerosis. Trends Pharmacol Sci 24:563–564CrossRefGoogle Scholar
  22. Nejadmoghaddam MR, Chamankhah M, Zarei S, Zarnani AH (2011) Profiling and quantitative evaluation of three nickel-coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation. J Nanobiotechnology 9:31CrossRefGoogle Scholar
  23. Rock KL, Kataoka H, Lai JJ (2013) Uric acid as a danger signal in gout and its comorbidities. Nat Rev Rheumatol 9:13–23CrossRefGoogle Scholar
  24. Rogers JK, Church GM (2016) Genetically encoded sensors enable real-time observation of metabolite production. Proc Natl Acad Sci U S A 113:2388–2393CrossRefGoogle Scholar
  25. Schallmey M, Frunzke J, Eggeling L, Marienhagen J (2014) Looking for the pick of the bunch: high-throughput screening of producing microorganisms with biosensors. Curr Opin Biotechnol 26:148–154CrossRefGoogle Scholar
  26. Shin HJ (2011) Genetically engineered microbial biosensors for in situ monitoring of environmental pollution. Appl Microbiol Biotechnol 89:867–877CrossRefGoogle Scholar
  27. Song SP, Wang LH, Li J, Zhao JL, Fan CH (2008) Aptamer-based biosensors. TrAC Trends Anal Chem 27:108–117CrossRefGoogle Scholar
  28. Tang SY, Cirino PC (2011) Design and application of a mevalonate-responsive regulatory protein. Angew Chem Int Ed 50:1084–1086CrossRefGoogle Scholar
  29. Tang SY, Fazelinia H, Cirino PC (2008) AraC regulatory protein mutants with altered effector specificity. J Am Chem Soc 130:5267–5271CrossRefGoogle Scholar
  30. Tang SY, Qian S, Akinterinwa O, Frei CS, Gredell JA, Cirino PC (2013) Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J Am Chem Soc 135:10099–10103CrossRefGoogle Scholar
  31. Teran W, Felipe A, Segura A, Rojas A, Ramos JL, Gallegos MT (2003) Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR. Antimicrob Agents Chemother 47:3067–3072CrossRefGoogle Scholar
  32. Toldra F, Reig M (2006) Methods for rapid detection of chemical and veterinary drug residues in animal foods. Trends Food Sci Technol 17:482–489CrossRefGoogle Scholar
  33. Vogel JR, Majewski MS, Capel PD (2008) Pesticides in rain in four agricultural watersheds in the United States. J Environ Qual 37:1101–1115CrossRefGoogle Scholar
  34. Wang QZ, Tang SY, Yang S (2017) Genetic biosensors for small-molecule products: design and applications in high-throughput screening. Front Chem Sci Eng 11:15–26CrossRefGoogle Scholar
  35. Wilkinson SP, Grove A (2004) HucR, a novel uric acid-responsive member of the MarR family of transcriptional regulators from Deinococcus radiodurans. J Biol Chem 279:51442–51450CrossRefGoogle Scholar
  36. Xiong DD, Lu SK, Wu JY, Liang CN, Wang W, Wang WZ, Jin JM, Tang SY (2017) Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor. Metab Eng 40:115–123CrossRefGoogle Scholar
  37. Yang D, Kim WJ, Yoo SM, Choi JH, Ha SH, Lee MH, Lee SY (2018) Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proc Natl Acad Sci U S A 115:9835–9844CrossRefGoogle Scholar
  38. Zhang C, Cui HY, Cai JR, Duan YQ, Liu Y (2015) Development of fluorescence sensing material based on CdSe/ZnS quantum dots and molecularly imprinted polymer for the detection of carbaryl in rice and chinese cabbage. J Agric Food Chem 63:4966–4972CrossRefGoogle Scholar
  39. Zuo X, Xiao Y, Plaxco KW (2009) High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices. J Am Chem Soc 131:6944–6945CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Beijing Key Laboratory of Plant Resources Research and DevelopmentBeijing Technology and Business UniversityBeijingChina

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