Abstract
Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction’s substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.
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Acknowledgments
Research was supported by EU 6th framework project Toxichip (http://www.toxichip.org) and by BARD grant number US-3864-06. We are grateful to O. Amster-Choder, The Hebrew University, Jerusalem, Israel and to R. A. LaRossa, DuPont Company, Wilmington, Delaware, USA for the gift of E. coli strains AG1688 and RFM443, respectively.
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Published in the special issue Microorganisms for Analysis with Guest Editor Gérald Thouand.
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Yagur-Kroll, S., Belkin, S. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon. Anal Bioanal Chem 400, 1071–1082 (2011). https://doi.org/10.1007/s00216-010-4266-7
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DOI: https://doi.org/10.1007/s00216-010-4266-7