Comparison of three genetically modified Escherichia coli biosensor strains for amperometric tetracycline measurement

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Abstract

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 μg ml−1 and 0.0026 μg ml−1, respectively, and their linear ranges were 0.1–1 μg ml−1 and 0–0.01 μg 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.

Highlights

▸ In this manuscript we report a genetically modified whole-cell based amperometric biosensor. ▸ The nuoA-based bioassays offered high sensitivity and specificity to the specific targets (tetracycline, copper and silver). ▸ This is the first time that a respiratory gene has been used as a reporter gene allowing the whole-cell sensor to directly quantify a target substrate by amperometric measurement of cell respiration responses.

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 commercial use to detect and quantify wastewater toxicity. It is a rapid catalytic microbial method in which the natural co-substrate, oxygen, is substituted by the mediator potassium ferricyanide (KFCIII) (Pasco et al., 2005). Electrons derived from oxidation of the substrate are released into the electron transport chain and ultimately to the external mediator KFCIII. This process leads to the accumulation of reduced mediator in solution. Transfer of electrons from the mediator to an electrode poised at a suitable voltage can generate 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 toxicants. 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 devised to achieve this objective. They were all based on gene induction in response to a specific toxicant. The antibiotic tetracycline (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 available 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 β-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 respiratory 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 reaction of serine to selenocysteine conversion on Ser-tRNASec using monoselenophosphate as the selenium donor (Allmang et al., 2009, Forchhammer and Bock, 1991, Forchhammer et al., 1991). Selenium 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).

In the third strategy, a key respiration gene was placed under the control of the tetA promoter such that, in the presence of Tet, the gene was expressed and the cells’ respiration rate increased. For this method, the nuoA gene, which encodes a protein that is directly involved in bacterial respiration, was used. The nuoA-N operon encodes the enzyme NADH dehydrogenase I, which catalyses the transfer of electrons from NADH into the start of the respiratory chain (Wackwitz et al., 1999), and functions in both anaerobic and aerobic conditions (Calhoun et al., 1993). The nuoA gene was fused to the Tn10 tetA promoter and then transformed into a nuoA knock-out E. coli mutant.

Section snippets

Materials and methods

Bacterial strains, plasmids and growth conditions.

The bacterial strains and plasmids used in this study are described in Table 1. All bacteria were E. coli strains cultured in shaking flask culture in LB medium at 200 rpm, 37 °C, and supplemented where appropriate, with: ampicillin (Amp) 100 μg ml−1, kanamycin (Kan) 50 μg ml−1, and Tet 10 μg ml−1.

Assay based on β-galactosidase expression

In order to convert the SciTox assay into a specific and more sensitive sensor, the E. coli biocomponent was re-engineered for Tet-induced expression of proteins that were expected to modify the SciTox response. In the first strategy, E. coli was transformed with the plasmid pSong8. E. coli (pSong8) expresses β-galactosidase in response to Tet. The expression of β-galactosidase allowed the cells to metabolize X-gal and produce a visible blue color. Two different growth phases were studied by

Conclusions

Three new SciTox biosensor strains were tested for their ability to quantify Tet. Two strains (E. coli (pSong8) and E. coli (pSong10)) responded specifically to Tet in a dose-dependent manner. The assays based on these 2 strains were considerably more sensitive than the unmodified SciTox assay and other similar DTA assays (Ertl et al., 2000) and may provide a strategy that could allow the SciTox assay to be developed as a sensitive and specific biosensor. However, the pSong9-based assay was not

Acknowledgment

This work was supported by funding from the New Zealand Foundation for Research, Science and Technology, contract LVLX0802.

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