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Analytical strategies for improving the robustness and reproducibility of bioluminescent microbial bioreporters

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Abstract

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions.

Schematic representation of the combined strategies to obtain a robust and reproducible whole-cell biosensor format: introduction of an internal viability control to correct the analyte-specific response, field-flow fractionation of bioreporters to obtain homogeneous cell populations and immobilization of fractionated bioreporters to preserve their viability.

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References

  1. Yagi K (2007) Applications of whole-cell bacterial sensors in biotechnology and environmental science. Appl Microbiol Biotechnol 73:1251–1258

    Article  CAS  Google Scholar 

  2. Eltzov E, Marks RS (2010) Whole-cell aquatic biosensors. Anal Bioanal Chem. doi:10.1007/s00216-010-4084-y

    Google Scholar 

  3. Chen A, Dietrich KN, Huo X, Ho SM (ANNO) developmental neurotoxicants in E-waste: an emerging health concern. Environ Health Perspect doi:10.1289/ehp.1002452

  4. Hynninen A, Virta M (2010) Whole-cell bioreporters for the detection of bioavailable metals. Adv Biochem Eng Biotechnol 118:31–63

    CAS  Google Scholar 

  5. Nivens DE, McKnight TE, Moser SA, Osbourn SJ, Simpson ML, Sayler GS (2004) Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. J Appl Microbiol 96:33–46

    Article  CAS  Google Scholar 

  6. Gu MB, Mitchell RJ, Kim BC (2004) Whole-cell-based biosensors for environmental biomonitoring and application. Adv Biochem Eng Biotechnol 87:269–305

    CAS  Google Scholar 

  7. Blouin K, Walker SG, Smit J, Turner R (1996) Characterization of in vivo reporter systems for gene expression and biosensor applications based on luxAB luciferase genes. Appl Environ Microbiol 62(6):2013–2021

    CAS  Google Scholar 

  8. Dorn JG, Frye RJ, Maier RM (2003) Effect of temperature, pH, and initial cell number on luxCDABE and nah gene expression during naphthalene and salicylate catabolism in the bioreporter organism Pseudomonas putida RB1353. Appl Environ Microbiol 69(4):2209–2216

    Article  CAS  Google Scholar 

  9. Neilson JW, Pierce SA, Maier RM (1999) Factors influencing expression of luxCDABE and nah genes in Pseudomonas putida RB1353(NAH7, pUTK9) in dynamic systems. Appl Environ Microbiol 65(8):3473–3482

    CAS  Google Scholar 

  10. Robbens J, Dardenne F, Devriese L, De Coen W, Blust R (2010) Escherichia coli as a bioreporter in ecotoxicology. Appl Microbiol Biotechnol 88(5):1007–1025

    Article  CAS  Google Scholar 

  11. Sørensen SJ, Burmølle M, Hansen LH (2006) Making bio-sense of toxicity: new developments in whole-cell biosensors. Curr Opin Biotechnol 17:11–16

    Article  Google Scholar 

  12. Hynninen A, Tõnismann K, Virta M (2010) Improving the sensitivity of bacterial bioreporters for heavy metals. Bioengineered Bugs 1(2):132–138

    Article  Google Scholar 

  13. Harms H, Wells MC, van der Meer JR (2006) Whole-cell living biosensors: are they ready for environmental application? Appl Microbiol Biotechnol 70:273–280

    Article  CAS  Google Scholar 

  14. Mirasoli M, Feliciano J, Michelini E, Daunert S, Roda A (2002) Internal response correction for fluorescent whole-cell biosensors. Anal Chem 74:5948–5953

    Article  CAS  Google Scholar 

  15. Reschiglian P, Zattoni A, Roda B, Michelini E, Roda A (2005) Field-flow fractionation and biotechnology. Trends Biotechnol 23:475–483

    Article  CAS  Google Scholar 

  16. Roda B, Zattoni A, Reschiglian P, Moon MH, Mirasoli M, Michelini E, Roda A (2009) Field-flow fractionation in bioanalysis. A review of recent trends. Anal Chim Acta 635:132–143

    Article  CAS  Google Scholar 

  17. Roda B, Reschiglian P, Zattoni A, Tazzari PL, Buzzi M, Ricci F, Bontadini A (2008) Human lymphocyte sorting by gravitational field-flow fractionation. Anal Bioanal Chem 392:137–145

    Article  CAS  Google Scholar 

  18. Roda B, Reschiglian P, Alviano F, Lanzoni G, Bagnara GP, Ricci F, Buzzi M, Tazzari PL, Pagliaro P, Michelini E, Roda A (2009) Gravitational field-flow fractionation of human hemopoietic stem cells. J Chromatogr A 1216:9081–9087

    Article  CAS  Google Scholar 

  19. Roda A, Cevenini L, Michelini E, Branchini BR (2011) A portable bioluminescence engineered cell-based biosensor for on-site applications. Biosens Bioelectron 26:3647–3653

    Article  CAS  Google Scholar 

  20. Branchini BR, Ablamsky DM, Davis AL, Southworth TL, Butler B, Fan F, Jathoul AP, Pule MA (2010) Red-emitting luciferases for bioluminescence reporter and imaging applications. Anal Biochem 396:290–297

    Article  CAS  Google Scholar 

  21. Sambrook EF, Fristch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  22. Hakkila K, Green T, Leskinen P, Ivask A, Marks R, Virta M (2004) Detection of bioavailable heavy metals in EILATox-Oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips. J Appl Toxicol 24:333–342

    Article  CAS  Google Scholar 

  23. Chung CT, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86:2172–2175

    Article  CAS  Google Scholar 

  24. Mascorro-Gallardo JO, Covarrubias AA, Gaxiola R (1996) Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae. Gene 172:169–170

    Article  CAS  Google Scholar 

  25. Gietz RD, Schiestl RH (1995) Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269

    Google Scholar 

  26. Belkin S, Gu MB (2010) Whole cell sensing systems. Advances in biochemical enginering. Springer, Heidelberg

    Book  Google Scholar 

  27. Ivask A, Rõlova T, Kahru A (2009) A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing. BMC Biotechnol 9:41

    Article  Google Scholar 

  28. Michelini E, Leskinen P, Virta M, Karp M, Roda A (2005) A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection. Biosens Bioelectron 20:2261–2267

    Article  CAS  Google Scholar 

  29. Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini BR, Roda A (2008) Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays. Photochem Photobiol Sci 7:212–217

    Article  CAS  Google Scholar 

  30. Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini BR, Roda A (2008) Spectral-resolved gene technology for multiplexed bioluminescence and high-content screening. Anal Chem 80:260–267

    Article  CAS  Google Scholar 

  31. Ivask A, Hakkila K, Virta M (2001) Detection of organomercurials with sensor bacteria. Anal Chem 73:5168–5171

    Article  CAS  Google Scholar 

  32. Council Directive 98/83/EC on the quality of water intended for human consumption as amended by Regulations 1882/2003/EC and 596/2009/EC

  33. Roda B, Lanzoni G, Alviano F, Zattoni A, Costa R, Di Carlo A, Marchionni C, Franchina M, Ricci F, Tazzari PL, Pagliaro P, Scalinci SZ, Bonsi L, Reschiglian P, Bagnara GP (2009) A novel stem cell tag-less sorting method. Stem Cell Rev 5:420–427

    Article  Google Scholar 

  34. Howlett NG, Avery SV (1999) Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae. FEMS Microbiol Lett 176:379–386

    Article  CAS  Google Scholar 

  35. Aksu Z, Egretli G, Kutsal A (1998) A comparative study of copper(II) biosorption on Ca-alginate, agarose and immobilized C. vulgaris in a packed-bed column. Process Biochem 33:393–400

    Article  CAS  Google Scholar 

  36. Jouanneau S, Durand MJ, Courcoux P, Blusseau T, Thouand G (2011) Improvement of the identification of four heavy metals in environmental samples by using predictive decision tree models coupled with a set of five bioluminescent bacteria. Environ Sci Technol 201145:2925–2931

    Article  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the financial support provided by the Italian Ministry of University and Research MIUR with the project FIRB 2009 “Development of bioremediation procedures and novel detection biotechnologies for Endocrine Disruptors” (prot. RBFR08SZTR), and the Istituto Superiore di Sanità (ISS) through the project “Progetto Strategico Salute della donna”. We are very grateful to Prof. Bruce Branchini for providing the cDNA encoding for PpyRE 8 and to Dr. Jorma Lampinen for the opportunity to test the Varioskan® Flash instrument. We thank Grace Fox for editing and proofreading the manuscript.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy

    Aldo Roda, Luca Cevenini, Elisa Michelini & Laura Mezzanotte

  2. INBB, Istituto Nazionale di Biostrutture e Biosistemi, Viale delle Medaglie d’Oro 305, 00136, Rome, Italy

    Aldo Roda, Barbara Roda, Elisa Michelini & Pierluigi Reschiglian

  3. Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126, Bologna, Italy

    Barbara Roda & Pierluigi Reschiglian

  4. Department of Food and Environmental Sciences, University of Helsinki, PO Box 56, 00014, Helsinki, Finland

    Marko Virta

  5. Department of Biochemistry and Food Chemistry, University of Turku, 20014, Turku, Finland

    Kaisa Hakkila

  6. Laboratory of Analytical and Bioanalytical Chemistry, Department of Pharmaceutical Sciences, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy

    Aldo Roda

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  1. Aldo Roda
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Correspondence to Aldo Roda.

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Fig. S1

Noncorrected dose–response curve for Cu2+ obtained with bacterial bioreporter that express PpyWT luciferase (solid line, circles) under the regulation of a copper-inducible promoter and the red-emitting luciferase PpyRE8 as internal viability control (dashed line, triangles) (S1a). Corrected dose–response curve for Cu2+ (S1b). Data are the average ± one standard deviation (n = 3) (PDF 187 kb)

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Roda, A., Roda, B., Cevenini, L. et al. Analytical strategies for improving the robustness and reproducibility of bioluminescent microbial bioreporters. Anal Bioanal Chem 401, 201–211 (2011). https://doi.org/10.1007/s00216-011-5091-3

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