DeVinney, RebekahLewenza, ShawnShideler, Steven Mark2019-09-302019-09-302019-09-19Shideler, S. M. (2019). Biosensors for the Detection of Naphthenic Acids in Wastewater from Oil Sands Operations (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/111101Naphthenic acids (NA) are a complex group of acyclic and cyclic alkyl-substituted carboxylic acids that are present in the bitumen mined from the oil sands. NAs accumulate in the tailings waste produced from processing bitumen, are toxic to living organisms and are difficult, both in terms of time and resources, to remediate. In this study, we established a high throughput pipeline using bacterial genomics and synthetic biology methods to build biosensor constructs using promoters from Pseudomonas synxantha, an organism isolated from oilsands process-affected water (OSPW). By observing the gene expression profiles of P. synxantha, we have been able to identify genes that are induced by NAs, and that likely play a role in the transport and catabolism of various NA species. We have identified a catabolic operon that is expressed in a dose-dependent manner, in response to NA exposure. In addition, we identified a TetR regulator that is divergent from this operon, that represses expression of the catabolic operon during normal conditions. The TetR regulator was purified and was shown to bind to the target promoter in electrophoretic mobility shift assays. In the presence of specific naphthenic acids, the repressor loses DNA binding affinity and no longer interacts with the promoter. Based on these findings, we have proposed a model of naphthenic acid sensing through a TetR repressor protein and have therefore identified all the components required to build an NA sensing biosensor using P. synxantha as a chassis. We have therefore identified all the components required to build an NA biosensor using P. synxantha as a chassis. The NA biosensor can be employed as a method to detect NA contamination in the environment, and also to aid in the discovery of novel genes for the purpose of supporting the bioremediation of the 1.2 trillion liters of NA contaminated water currently being stored in Northern Alberta.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.biosensorsnaphthenic acidssynthetic biologyBioinformaticsGeneticsMicrobiologymaster thesis10.11575/PRISM/37162