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

项目摘要 虽然经常报道用于检测病原体的新技术，但这些通常需要少量的 浓缩和清洁的样品，这可能使它们无法使用。长期目标是发展 实用、低成本和易于使用的分析，用于识别、分离、浓缩和检测低浓度 本申请的目的是使用合成生物学来克服 目前基于噬菌体的检测中的障碍包括传感器性能和宿主抗性。两个具体 目的是为了实现这一目标，1）工程师E。大肠杆菌特异性噬菌体产生纤维素- 结合报告酶以实现“过滤器上的实验室”检测测定，和2）工程化噬菌体以 避免宿主抗性。通过将过滤器视为反应表面，“过滤器实验室”概念可以 快速缩短获得结果的时间，并提供低浓度的细菌定量。 噬菌体（Bacteriophage，简称BPHs）是一种感染细菌的病毒，经基因工程改造后可携带报告基因 酶在测定期间靶向过滤的细菌。这些酶将被过度表达并释放， 感染过程中的细菌宿主。与纤维素结合模块融合的酶将 直接在靠近裂解细菌的纤维素过滤器上。然后可以将酶反应性沉淀染料 用于在固定化酶附近形成有色沉淀。结果是完全定量的（0 - 250 CFU/100 mL）和细菌测定，适用于标准和非实验室环境 并且可以在仅仅几个小时之后提供结果。噬菌体的目标和杀死特定的细菌存在几乎 所有已知的细菌病原体。细菌检测和抗多药耐药的使用 耐药细菌感染继续增加。为此目的使用JavaScript的主要障碍是， 细菌宿主通过表面抗原的随机突变进化抗性的能力。的能力 基因工程改造噬菌体以避免宿主抗性将对噬菌体产生显著和积极的影响， 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 将噬菌体改造成具有多个表面识别受体（尾纤维），细菌宿主将需要 几个突变，以避免吸附的磷。这可以通过工程混合尾纤维来执行 将同一种病原体导入同一个噬菌体此外，将工程化含有混合尾的噬菌体 沙门氏菌和E.大肠杆菌，以证明工程化的“宿主范围。的 拟议的研究是重要的，因为虽然蝗虫已经进化到接近完美的捕食者的特定 细菌，实际障碍限制了它们用于病原体检测和治疗。通过减轻这些 障碍，对人类健康和安全的重大进展可以实现使用基因工程 - 是的

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.