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受油砂工艺影响的水(OSPW)中存在许多污染物。市政水处理厂已经确定了几种未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特性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，不仅应用于直接水质检测，而且还用于识别生物修复基因。 目的1.构建、验证和优化检测重要水污染物的全生物传感器。 生物传感器是一种工程细菌菌株，可以专门检测低水平的小分子，并产生简单、定量的输出。对分析物作出反应的启动子被克隆为LuxCDABE报告基因的转录融合。OSPW的优先目标是环烷酸(NA)和多环芳烃(PAHs)，这些物质丰富且有毒。卡马西平(抗癫痫药)和双氯芬酸(抗炎药)是城市水处理后仍留在污水中的药物。我们从OSPW中回收了假单胞菌，并对4株假单胞菌的基因组草稿进行了测序。生物传感器底盘菌株为尾矿分离株。我们利用细菌基因组学和合成生物学建立了一条管道，可以快速创造出对任何环境污染物都适用的生物传感器菌株。RNA-SEQ用于鉴定污染物暴露诱导的细菌基因/启动子。从诱导基因中合成了84个优先启动子，并克隆为转录LUX融合基因。将对质粒编码的生物传感器电路进行剂量依赖的基因表达、灵敏度和特异性测试。我们将探索代谢物感应转录抑制物对污染物的感应机制，并将其纳入生物传感器的改进设计中。 目的2.筛选元基因组文库，确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推广的技术，在治疗OSPW方面具有潜在的应用前景。尾矿库微生物群落的多样性无法培养，但我们可以通过筛选生物修复基因的元基因组环境DNA(EDNA)来利用这种遗传多样性。我们优化了在污染物存在的情况下培养大肠杆菌宿主的共培养系统，然后在384个孔型实验中共培养我们的假单胞菌生物传感器，以鉴定降解污染物的菌株。将用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的机制，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.