Bacterial biosensors for detection and bioremediation of wastewater pollutants.

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Numerous pollutants are present in the oilsands process-affected water (OSPW) generated from hot water extraction of bitumen from the Athabasca oilsands. Municipal water treatment plants have identified several pharmaceutical drugs that are not completely removed and are released in the effluent. The high throughput and inexpensive nature of biosensors can support long-term environmental testing programs. We propose to develop a biosensor technology for application in direct water testing, but also for identifying bioremediation genes. Aim 1. To construct, validate and optimize whole biosensors for important water pollutants. Biosensors are engineered bacterial strains that can specifically detect low levels of small molecules and produce a simple, quantitative output. Promoters responsive to an analyte are cloned as a transcriptional fusion to the luxCDABE reporter genes. The priority OSPW targets are naphthenic acids (NA) and polycyclic aromatic hydrocarbons (PAHs), which are abundant and toxic. Carbamazepine (anti-epileptic) and diclofenac (anti-inflammatory) are pharmaceuticals that remain in the effluent after municipal water treatment. We recovered Pseudomonas isolates from OSPW and sequenced the draft genomes of 4 strains. The P. synxantha tailings isolate was used as the biosensor chassis strain. We established a pipeline using bacterial genomics and synthetic biology to rapidly create biosensor strains for any environmental pollutant. RNA-seq was performed to identify bacterial genes/promoters induced in response to pollutant exposure. Eighty-four priority promoters from induced genes were synthesized and cloned as transcriptional lux fusions. Plasmid-encoded biosensor circuits will be tested for dose-dependent gene expression, sensitivity and specificity. The mechanism of pollutant sensing by metabolite-sensing transcriptional repressors will be explored and incorporated into an improved design of biosensors. Aim 2. To screen metagenomic libraries and identify bioremediation genes for target pollutants. Bioremediation is a cost-effective and scalable technology for potential use in treating OSPW. The diverse community of tailings ponds microbes cannot be grown, but we can exploit this genetic diversity by screening metagenomic environmental DNA (eDNA) for bioremediation genes. We optimized a co-culture system of growing the E. coli host in the presence of the pollutant, and then co-culturing our Pseudomonas biosensor in 384 well format experiments, to identify strains that degrade the contaminant. Degradation will be confirmed with GC-MS and plasmids from positive clones will be sequenced. The specific gene function will be confirmed by bioinformatic, mutation and phenotype analyses. The genetic information gained in these studies will help elucidate the mechanisms of degradation, and will support synthetic biology strategies to construct engineered microbes with improved bioremediation.