Biosensors and Bioelectronics 35 (2012) 69–74 Contents lists available at SciVerse ScienceDirect Biosensors and Bioelectronics j our na l ho me page: www.elsevier.com/locate/bios Comparison of three genetically modified Escherichia coli biosensor strains for amperometric tetracycline measurement Wenfeng Song a,∗ , Neil Pasco b , Ravi Gooneratne a , Richard J. Weld b a Lincoln University, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand b Lincoln Ventures Limited, P.O. Box 133, Lincoln University, Lincoln 7647, New Zealand a r t i c l e i n f o Article history: Received 30 November 2011 Received in revised form 5 February 2012 Accepted 6 February 2012 Available online 3 March 2012 Keywords: Biosensor lacZ, nuoA Tetracycline Copper Silver a b s t r a c t Three separate genetic strategies, based upon the induced expression of three different genes (lacZ, selA and nuoA) were tested to provide the SciTox assay with sensitive and specific detection of the antibiotic tetracycline (Tet). All three strategies relied on gene induction from the Tn10 tetA promoter. Both lacZ and nuoA biosensors responded specifically and sensitively to sub-inhibitory concentrations of Tet. However, the selA-based assay was not sensitive enough to detect Tet in the SciTox assay. The detection limits for Tet of the lacZ and nuoA biosensor strains were 0.11 g ml -1 and 0.0026 g ml -1 , respectively, and their linear ranges were 0.1–1 g ml -1 and 0–0.01 g ml -1 , respectively. While lacZ has previously been used as a reporter gene in an amperometric bioassay, nuoA is a novel and more sensitive reporter gene. This is the first report in which a respiratory gene was used as a reporter gene in an amperometric biosensor. The results indicate that this approach can produce a highly sensitive detection system. In order to test whether the new system could be used to detect other chemicals, the nuoA gene was re-engineered to be driven by the copper-inducible copA promoter. Using this strain, the SciTox assay was found to be able to specifically detect copper and silver ions. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The SciTox toxicity assay is a commercially available, whole cell microbial assay that measures toxicity through inhibition of bacterial respiration (Tizzard et al., 2004) and is currently in com- mercial use to detect and quantify wastewater toxicity. It is a rapid catalytic microbial method in which the natural co-substrate, oxy- gen, is substituted by the mediator potassium ferricyanide (KFCIII) (Pasco et al., 2005). Electrons derived from oxidation of the sub- strate are released into the electron transport chain and ultimately to the external mediator KFCIII. This process leads to the accumu- lation of reduced mediator in solution. Transfer of electrons from the mediator to an electrode poised at a suitable voltage can gen- erate a measurable current and is used to quantify the magnitude of respiration inhibition and indirectly the toxicity. In its current configuration, the Scitox assay measures toxicity nonspecifically and therefore cannot distinguish between toxi- cants. In order to create a bio-assay that can identify and quantify specific toxicants at concentrations lower than inhibitory levels, the SciTox assay was re-engineered. Three separate strategies were ∗ Corresponding author. Tel.: +64 3 325 3700; fax: +64 3 325 3725. E-mail addresses: Songw2@hotmail.com (W. Song), pascon@lvl.co.nz (N. Pasco), Ravi.Gooneratne@lincoln.ac.nz (R. Gooneratne), weldr@lvl.co.nz (R.J. Weld). devised to achieve this objective. They were all based on gene induction in response to a specific toxicant. The antibiotic tetra- cycline (Tet) and the Tn10 tetA promoter, which is de-repressed in response to the presence of Tet, were used as the model system. In the first strategy, the quantity of metabolisable carbon avail- able to the bacteria in the SciTox assay was manipulated by a method used previously in yeast (Lehmann et al., 2000; Tag et al., 2007). In this method, lactose is used as the sole carbon source for bacteria in the SciTox assay. Escherichia coli strains lacking the lacZ gene do not produce -galactosidase and are incapable of utilizing lactose as a carbon source. By re-introducing the lacZ gene fused to the tetA promoter into these cells, metabolisable carbon is only available to the cells in the presence of Tet. In the second strategy, the overall redox activity of the respi- ratory enzymes was manipulated. For this method, the selA gene, which encodes the enzyme selenocysteine synthase (Tormay et al., 1998), was fused to the tetA promoter and transformed into a selA knock-out E. coli mutant. The selA gene catalyzes the reac- tion of serine to selenocysteine conversion on Ser-tRNA Sec using monoselenophosphate as the selenium donor (Allmang et al., 2009; Forchhammer and Bock, 1991; Forchhammer et al., 1991). Sele- nium atoms have similar properties to sulfur atoms. However, using Selenium instead of sulfur, the catalytic rate of seleno-proteins is significantly increased (Tormay et al., 1998). 0956-5663/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2012.02.023