Bacterial biosensors for detection and bioremediation of wastewater pollutants.

从阿萨巴斯卡油砂中热水提取沥青产生的油砂工艺影响水（OSPW）中存在许多污染物。市政水处理厂已经确定了几种没有完全去除并在污水中释放的药物。生物传感器的高通量和廉价的性质可以支持长期的环境测试计划。我们建议开发一种生物传感器技术，应用于直接水测试，但也用于识别生物修复基因。* 目标1。构建、验证和优化重要水污染物的整体生物传感器。生物传感器是经过工程改造的细菌菌株，可以特异性地检测低水平的小分子，并产生简单的定量输出。将响应于分析物的启动子克隆为与luxCDABE报告基因的转录融合物。OSPW的优先目标是环烷酸（NA）和多环芳烃（PAH），这是丰富的和有毒的。卡马西平（抗癫痫药）和双氯芬酸（抗炎药）是市政水处理后残留在污水中的药物。我们从OSPW中回收假单胞菌分离株，并对4种菌株的基因组草图进行测序。将合黄单胞菌尾矿分离物用作生物传感器底盘菌株。我们利用细菌基因组学和合成生物学建立了一个管道，可以快速创建针对任何环境污染物的生物传感器菌株。进行RNA-seq以鉴定响应于污染物暴露而诱导的细菌基因/启动子。从诱导基因的84个优先启动子的合成和克隆作为转录lux融合。将测试质粒编码的生物传感器电路的剂量依赖性基因表达、灵敏度和特异性。将探索通过代谢物感应转录阻遏物感应污染物的机制，并将其纳入生物传感器的改进设计中。目标2.筛选宏基因组文库并鉴定目标污染物的生物修复基因。生物修复是一种具有成本效益和可扩展性的技术，可用于处理OSPW。尾矿池微生物的多样性社区无法生长，但我们可以通过筛选宏基因组环境DNA（eDNA）的生物修复基因来利用这种遗传多样性。我们优化了一个生长E.大肠杆菌宿主在污染物的存在下，然后共培养我们的假单胞菌生物传感器在384孔格式的实验，以确定菌株降解污染物。将采用GC-MS确认降解，并对阳性克隆的质粒进行测序。将通过生物信息学、突变和表型分析确认特定基因功能。在这些研究中获得的遗传信息将有助于阐明降解机制，并将支持合成生物学策略，以构建具有改进的生物修复的工程微生物。

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.**