Modification of a whole-cell biosensor to detect toxins by SciToxTM rapid DTA assay

Abstract

Pollution of soil and water caused by human activity such as agriculture and industry is an issue of great global concern. In order to mitigate, eliminate or avoid pollution it is necessary to be able to accurately measure and monitor it. Therefore, rapid and sensitive methods which can detect and quantify environmental toxicants are being developed. The SciToxTM rapid mediated direct toxicity assessment (DTA) assay was developed by Lincoln Ventures Limited. It is a rapid catalytic microbial method in which the natural co-substrate, oxygen, is substituted by a synthetic co-substrate or mediator. 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. The SciToxTM assay has two major constraints; the stability of whole cells and a lack of specificity to a target analyte. These two aspects are addressed in this thesis. Part A: Three microorganisms, Acinetobacter calcoaceticus (A. calcoaceticus), Escherichia coli (E. coli) and Pseudomonas putida (P. putida) were evaluated as biocomponents in a whole-cell based bio-assay. Each of these bacteria was prepared in two forms, as freshly cultured cells and as freeze dried cells. In order to compare the storage efficiency of different cryoprotectants, each freeze-dried bacterial strain was prepared in two forms, pre-treated with either polyethylene glycol (PEG) or sucrose / Tween 80, and stored at two different temperatures, 4 ˚C and -20 ˚C. This was to validate that, after optimum freeze dried storage conditions, freeze-dried cells exhibited a similar response to the standard toxicants, 2,4-dichlorophenol (2,4-DCP) and 3,5-dichlorophenol (3,5-DCP) as their freshly cultured counterparts. In the freeze dried cell storage study, sucrose / Tween 80 was more efficient in maintaining the cell viability. In contrast, PEG was more suitable for SciToxTM toxicity assay because it produced cells with more conventional EC50 values (closer to fresh ones). After one and two-month storage at either 4 ˚C or -20 ˚C, the viability of the cells decreased significantly but showed a similar dose-response curve, when tested in the DTA SciToxTM assay following a protocol identical to that applied to the fresh cell counterparts. After three-month stored at -20 ˚C, all three freeze-dried strains could be used to assay the toxicity of standard toxicants but at 4 ˚C, A. calcoaceticus and E. coli lost their activity in SciToxTM assay. Bacterium P. putida was the most resistant to freeze-drying and subsequent storage, and also the most stable strain in SciToxTM DTA assay compared with the other two. This is the first time the shelf-life of freeze-dried cells has been evaluated in a commercial toxicity assay. Part B: The objective was to develop a biosensor strain of E. coli that is specific for detecting and quantifying antibiotics in milk samples by genetically engineering the bacterial component of the SciToxTM assay. The SciToxTM assay in its current configuration, 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 SciToxTM assay was re-engineered by using specific promoters and reporter gene fusions for gene induction in response to a specific toxicant. Four separate strategies were investigated using four different reporter genes, lacZ, selA, ompF and nuoA. All strategies were based on gene induction in response to a specific toxicant. The Tn10 tetA promoter, de-repressed in response to the presence of tetracycline (Tet), and the copA promoter, activated in the response to the presence of copper (Cu) and silver (Ag), were used as the model systems. In each reporter system, the presence of Tet (tetA promoter regulated systems) or the presence of Cu and/or Ag (copA promoter regulated systems) led to increased expression of the reporter gene which produced an increase in respiratory activity and therefore an increased SciToxTM signal. In conclusion, the three microorganisms, A. calcoaceticus, E. coli and P. putida could be freeze-dried and stored at -20 ˚C for up to 3 months and still be useful in the SciToxTM assay. Results of re-engineering the SciToxTM assay suggested that two genes (the ompF and the nuoA) could be used as reporter genes to improve the sensitivity and specificity of SciToxTM DTA toxicity assay. To our knowledge, this is the first time nuoA gene, or any other respiratory gene, has been used as the reporter gene for an amperometric biosensor.