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

用于病原体分离、浓缩和检测的噬菌体实验室过滤器

• 批准号: 9762099
• 负责人: Sam R Nugen
• 金额: $ 18.73万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762099
• 关键词: Address; Adopted; Adsorption; Affinity; Antigens; Bacteria; Bacterial Infections; Bacteriophage T4; Bacteriophages; Binding; Biological Assay; Biosensor; Carbohydrates; Cellular Phone; Cellulose; Clinical; Colony-forming units; Color; Combating Antibiotic Resistant Bacteria; Custom; Cytolysis; DNA; Detection; Development; Diagnostic; Discrimination; Distal; Dyes; Engineering; Enzyme Reactivation; Enzymes; Equipment; Escherichia coli; Escherichia coli O157:H7; Fiber; Genes; Genetic Engineering; Goals; Health; Host resistance; Hour; Human; Immobilization; Immobilized Enzymes; Infection; Knowledge; Lead; Life Cycle Stages; Liquid substance; Location; Magnetism; Mediating; Methods; Multi-Drug Resistance; Mutation; Nanotechnology; Optics; Outcome; Pathogen detection; Performance; Precipitation; Preparation; Process; Protocols documentation; Reaction; Reporter; Reporter Genes; Reporting; Research; Resistance; Safety; Salmonella; Sampling; Sodium Chloride; Stains; Structural Protein; Surface; Surface Antigens; Tail; Techniques; Technology; Temperature; Time; Virus; Visual; Water; Work; base; combat; cost; design; diagnostic assay; medical food; meetings; multiplex detection; new technology; overexpression; pathogen; pathogenic bacteria; protein structure; rapid detection; receptor; sensor; synthetic biology; tool

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.

项目概要 虽然经常报道检测病原体的新技术，但这些技术通常需要少量的 浓缩且干净的样品可能使其无法实际使用。长期目标是发展 实用、低成本且易于使用的检测方法，用于识别、分离、浓缩和检测低浓度 该应用的目的是利用合成生物学来克服液体样品中目标细菌的 当前基于噬菌体的检测的障碍包括传感器性能和宿主电阻。两个具体 我们已针对这一目标制定了目标，1）设计大肠杆菌特异性噬菌体来生产纤维素- 结合报告酶以实现“过滤器实验室”检测分析，以及 2) 工程噬菌体 避免宿主抵抗。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细菌定量。 噬菌体（噬菌体）是感染细菌的病毒，可以被改造为报告基因传递基因 在测定过程中针对过滤后的细菌的酶。这些酶将被过度表达并释放 感染期间的细菌宿主。与纤维素结合模块融合的酶将固定 直接在靠近裂解细菌的纤维素过滤器上。然后可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）和细菌检测，适用于标准和非实验室设置 只需几个小时即可提供结果。针对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。使用噬菌体检测细菌和对抗多种药物 耐药细菌感染持续增加。使用噬菌体达到此目的的主要障碍是 细菌宿主通过表面抗原的随机突变进化出耐药性的能力。有能力 对噬菌体进行基因改造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。经过 改造噬菌体使其具有多个表面识别受体（尾纤维），细菌宿主需要 一些突变以避免噬菌体的吸附。这可以通过设计混合尾纤来实现 将相同的病原体靶向一个噬菌体。此外，还将设计出包含混合尾部的噬菌体 沙门氏菌和大肠杆菌特有的纤维，用于展示噬菌体宿主范围的工程设计。这 拟议的研究具有重要意义，因为虽然噬菌体已进化为近乎完美的特定捕食者 细菌，实际障碍限制了它们在病原体检测和治疗中的应用。通过减轻这些 克服障碍，利用基因工程可以在人类健康和安全方面取得重大进展 噬菌体。