Bacterial biosensors for detection and bioremediation of wastewater pollutants.

从阿萨巴斯卡油砂中热水提取沥青所产生的油砂工艺影响水 (OSPW) 中存在多种污染物。市政水处理厂已发现几种未完全去除并释放到废水中的药物。生物传感器的高通量和廉价特性可以支持长期的环境测试项目。我们建议开发一种生物传感器技术，用于直接水质检测，也可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，可以特异性检测低水平的小分子并产生简单的定量输出。对分析物有反应的启动子被克隆为 luxCDABE 报告基因的转录融合体。 OSPW 的优先目标是环烷酸 (NA) 和多环芳烃 (PAH)，它们含量丰富且有毒。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废水中的药物。我们从 OSPW 中回收了假单胞菌分离株，并对 4 个菌株的基因组草案进行了测序。 P. synxantha 尾矿分离株用作生物传感器底盘菌株。我们建立了利用细菌基因组学和合成生物学的管道，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 来鉴定因污染物暴露而诱导的细菌基因/启动子。来自诱导基因的 84 个优先启动子被合成并克隆为转录 lux 融合体。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、敏感性和特异性。将探索代谢物传感转录抑制因子的污染物传感机制，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且可扩展的技术，可用于治疗 OSPW。尾矿池微生物的多样化群落无法生长，但我们可以通过筛选宏基因组环境 DNA (eDNA) 中的生物修复基因来利用这种遗传多样性。我们优化了共培养系统，在存在污染物的情况下培养大肠杆菌宿主，然后在 384 孔格式实验中共培养我们的假单胞菌生物传感器，以识别降解污染物的菌株。将通过 GC-MS 确认降解，并对阳性克隆的质粒进行测序。具体的基因功能将通过生物信息学、突变和表型分析来确认。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来构建具有改进生物修复功能的工程微生物。

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.