Phage-Enabled Lab-on-a-Filter for Pathogen Separation, Concentration, and Detection

项目摘要 虽然检测病原体的新技术经常被报道，但这些技术通常需要少量的 浓缩和清洁的样品会使其不切实际地使用。我们的长远目标是发展 实用、低成本和易于使用的分析方法，用于识别、分离、浓缩和检测低浓度 液体样本中的目标细菌这一应用的目的是使用合成生物学来克服 目前基于噬菌体检测的障碍包括传感器性能和宿主抵抗力。两个具体的 朝着这个目标发展的目标是，1)设计一种大肠杆菌特异的噬菌体来生产一种纤维素- 结合报告酶，以实现“Lab on a Filter”检测分析，以及2)工程噬菌体，以 避免寄主抗性。通过将过滤器视为反应面，可以对过滤器进行实验的概念 快速缩短结果的时间，并提供低浓度的细菌定量。 噬菌体是感染细菌的病毒，可以通过基因工程为报道者传递基因。 在化验过程中以过滤的细菌为靶标的酶。这些酶会被过度表达和释放 感染过程中的细菌宿主。与纤维素结合模块融合的酶将固定化 直接放在裂解细菌附近的纤维素过滤器上。然后酶活性沉淀染料就可以 用于在固定化酶附近形成有色沉淀物。结果是一个完全定量的(0 -250 cfu/100毫升)和细菌检测，符合标准和非实验室设置 并且只需几个小时就可以提供结果。以特定细菌为靶标并杀死特定细菌的噬菌体几乎存在 所有已知的细菌病原体。噬菌体在细菌检测和对抗多药中的应用 抗药性细菌感染继续增加。将噬菌体用于此目的的主要障碍是 细菌宿主通过表面抗原的随机突变而进化出抗药性的能力。有能力 对噬菌体进行基因工程以避免宿主抗性将对噬菌体产生重大而积极的影响- 基于病原体检测以及噬菌体疗法来治疗多重耐药细菌感染。通过 将噬菌体设计成具有多个表面识别受体(尾部纤维)，细菌宿主将需要 几个突变以避免噬菌体的吸附。这可以通过工程混合尾部纤维来实现 将相同的病原体定位到一个噬菌体中。此外，还将设计一种含有混合尾巴的噬菌体 沙门氏菌和大肠杆菌特有的纤维，以展示噬菌体宿主范围的工程。这个 拟议中的研究意义重大，因为虽然噬菌体已经进化成近乎完美的捕食者，但它们是特定的 在细菌方面，实际障碍限制了它们在病原体检测和治疗方面的使用。通过缓解这些问题 障碍，人类健康和安全的重大进步可以通过使用基因工程来实现 噬菌体。

Project Summary While new technologies for detecting pathogens are often reported, these typically require small volumes of concentrated and clean samples which can make them impractical to use. The long-term goal is to develop pragmatic, low-cost and easy-to-use assays to identify, separate, concentrate, and detect low concentrations of target bacteria in liquid samples The objective of this application is to use synthetic biology to overcome current obstacles in phage-based detection including sensor performance and host resistance. Two specific aims have been developed towards this objective, 1) Engineer an E. coli-specific phage to produce a cellulose- binding reporter enzyme to enable a “Lab on a Filter” detection assay, and 2) Engineering bacteriophages to avoid host resistance. By considering a filter to be a reaction surface, a “Lab on a Filter” concept which can rapidly reduce the time to results and provide low concentration quantification of bacteria in enabled. Bacteriophages (phages) are viruses which infect bacteria, and can be engineered to deliver genes for reporter enzymes to target filtered bacteria during an assay. The enzymes would be overexpressed and released by the bacterial host during the infection. Enzymes fused with a cellulose-binding module would immobilize directly on a cellulose filter in proximity to the lysed bacteria. Enzyme-reactive precipitating dyes can then be used to form colored precipitate in the proximity of the immobilized enzymes. The result is a fully quantitative (0 – 250 CFU/100 mL) and assay for bacteria which is amenable to both standard and non-laboratory settings and can be provide results after only a few hours. Phages which target and kill specific bacteria exist for almost all known bacterial pathogens. The use of phages for both bacteria detection and for combating multidrug resistant bacterial infections continues to increase. The main hurdle with using phages for this purpose, is the ability of the bacterial host to evolve resistance through random mutations of surface antigens. The ability to genetically engineer phages to avoid host resistance will have a significant and positive impact on phage- based pathogen detection as well as phage therapy to treat multidrug-resistant-bacterial infections. By engineering a phage to have multiple surface recognition receptors (tail fibers), the bacterial host would require several mutations to avoid adsorption of the phages. This can be performed by engineering mixed tail fibers targeting the same pathogen into one phage. In addition, a phage will be engineered that contains mixed tail fibers specific to Salmonella and E. coli to demonstrate the engineering of the phages' host range. The proposed research is significant because while phages have evolved to be near perfect predators of specific bacteria, practical hurdles have limited their use for pathogen detection and treatment. By mitigating these hurdles, significant advances toward human health and safety can be achieved using genetically engineered phages.