Monitoring aromatic hydrocarbons by whole cell electrochemical biosensors

doi:10.1016/j.ab.2004.08.032

Analytical Biochemistry

Volume 335, Issue 2, 15 December 2004, Pages 175-183

https://doi.org/10.1016/j.ab.2004.08.032 Get rights and content

Abstract

In this article, we describe a bacterial whole cell electrochemical biosensors system that can be used for monitoring aromatic hydrocarbons. These bacterial biosensors are based on fusions of a promoter that is sensitive to aromatic compounds (the promoter region of the xylS gene and the xylR gene coding for the transcriptional regulator of the xyl operon) to reporter genes that can be monitored electrochemically at real-time and on-line. The xylS promoter was fused upstream of two promoterless genes coding the lacZ gene and phoA. These constructs reacted specifically to aromatic compounds but not to nonaromatic compounds, and we could detect, within minutes, micromolar concentrations of different aromatic hydrocarbons such as xylene and toluene. The use of two different reporter genes allows the future construction of a multianalyte detection system for simultaneous monitoring of several pollutants. These whole cell biosensors are potentially useful for on-line and in situ detection of aromatic compounds and as early warning systems of environmental hazards.

Section snippets

Reagents

PAPG, ampicillin, X-gal (5-bromo-4-chloro-3-indolyl-β-galactoside), X-pho (5-bromo-4-chloro-3-indolyl phosphate, BCIP), and all aromatic and nonaromatic hydrocarbons were purchased from Sigma (Israel). Luria–Bertani (LB) broth and agar were purchased from Difco Becton Dickinson (USA). PAPP was self-synthesized. Restriction endonucleases and T4-DNA ligase were purchased from New England Biolab (USA). Screen print disposable electrodes, consisting of a carbon working electrode (3.14 mm²), a carbon

Construction and specific induction of the biosensors strains

The construction of plasmid pXylRS–LacZ was described in Materials and methods. In addition, we constructed a second plasmid, pXylRS–AP (also described in Materials and methods), which is similar to pXylRS–LacZ but contains the phoA gene as a reporter gene. Essentially, both constructs contain the reporter gene (lacZ or phoA) under the control of the xylS promoter. The plasmids were transformed into MC1061 cells, forming the biosensor strains MC1061–pXylRS–LacZ and MC1061–pXylRS–AP.

The

Discussion

In this article, we have described the characterization and testing of two electrochemical biosensors for the detection of toluene and related compounds. These are based on placing reporter genes, coding for enzymes with electrochemically detected activity, under the control of the xyl operon. The biosensors are fast reacting, specific to aromatic compounds, quantitative, and sensitive (detecting even vapor of benzene and toluene). The results and the simplicity of the measurements suggest the

Acknowledgments

We thank Tova Neufeld for her help and the sequencing unit at Tel Aviv University. We are grateful to the Peikovsky Valachi grant to Yossi Paitan. This work was supported by the Israel Ministry of Science and Technology and by the Manja and Morris Leigh Chair for Biophysics and Biotechnology (Eliora Z. Ron).

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Monitoring aromatic hydrocarbons by whole cell electrochemical biosensors

Abstract

Section snippets

Reagents

Construction and specific induction of the biosensors strains

Discussion

Acknowledgments

Gene

Biosens. Bioelectron.

Trends Biotechnol.

Trends Biotechnol.

Gene

Biotechnol. Adv.

J. Microbiol. Methods

J. Fresenius Anal. Chem.

Chem. Rev.

EXS

Anal. Chem.

Microbiology

Environ. Microbiol.

Appl. Environ. Microbiol.

Appl. Environ. Microbiol.