Metabolite-Sensing Transcription Factors for Developing Whole Cell Naphthenic Acid Biosensor Technology
Date
2022-09
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Naphthenic Acids (NA) are a complex group of acyclic and cyclic alkyl-substituted carboxylic acids. These compounds are naturally produced during the degradation of petroleum and are present in high concentrations in the waste produced during the bitumen extraction process. NAs are very toxic, and monitoring and treating the oil sands process-affected water (OSPW) is costly and time-consuming. Whole cell biosensors are a proven technology holding the potential for a rapid and low-cost method of monitoring NA in tailings ponds. We are exploiting the sensitive and reliable detection mechanisms present in bacterial cells for inducing appropriate responses to environmental changes, by engineering bacterial strains to produce a simple and quantitative output in proportion to NA. Using bacterial genomics and synthetic biology, we designed transcription factor-based biosensors using a novel Pseudomonas species isolated from the OSPW, by examining the upregulated genes in the presence of different NA mixtures. The promoters of these genes are then synthesized and cloned directly upstream from a bacterial lux operon, that is used as a bioluminescent reporter. We are particularly interested in those involved in transcriptional regulation, antibiotic efflux, or bioremediation. We have identified three strong promoters for NA-detection: a promoter driving the expression of a hypothetical gene, a divergent NA-inducible promoter from the atu operon regulated by a TetR family repressor, and a promoter for a toxic RND efflux system regulated by a MarR family regulator. These transcriptional regulators bind within the promoter sequence to repress gene expression, unless in the presence of a target analyte. Upon analyte binding, a confirmational change occurs to release the protein from the promoter region and allow transcription. Using electrophoretic mobility shift assays, this mechanism of promoter repression and NA detection was confirmed for both AtuR and MarR regulators. Through lux expression experiments, the hypothetical promoter, divergent atu promoter, and MarR regulated promoter were confirmed to sensitively respond in a dose-dependent manner to a custom mixture of NA compounds, a mixture of primarily acyclic NAs, and the complex OSPW mixture, respectively. These biosensors are sensitive within 2-30 parts per million, and the capability of these biosensors to uniquely respond to NA compounds and not other hydrocarbons such as alkanes and BTEX, demonstrates the potential for using a panel of these biosensors for detecting NA toxicity in the environment.
Description
Keywords
Biosensor, Tailings ponds, Naphthenic Acids, Transcription factors, Environmental Monitoring
Citation
Bookout, T. (2022). Metabolite-sensing transcription factors for developing whole cell naphthenic acid biosensor technology (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.