Bioengineering Phage-based Biosensors with Genetic Specificity and High Sensitivity

具有遗传特异性和高灵敏度的生物工程噬菌体生物传感器

• 批准号: 10727412
• 负责人: Sam R Nugen
• 金额: $ 18.78万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727412
• 关键词: Abate; Address; Alkaline Phosphatase; Animals; Antibiotic Resistance; Bacteria; Bacterial Genome; Bacterial Infections; Bacteriophage T4; Bacteriophages; Binding; Biological Assay; Biomedical Engineering; Biosensing Techniques; Biosensor; Cells; Cessation of life; Complement; Cytolysis; DNA; Dedications; Detection; Development; Diagnostic; Elements; Engineering; Environmental Protection; Enzymes; Equipment; Escherichia coli; Exposure to; Food Safety; Gene Deletion; Gene Silencing; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome; Goals; Green Fluorescent Proteins; Health; Human; Infection; Laboratories; Luciferases; Magnetism; Medicine; Metabolic; Methods; Outcome; Pathogenicity; Phase; Plasmids; Polymerase; Proteins; Reagent; Refrigeration; Reporter; Research; Resource-limited setting; Resources; Sample Size; Sampling; Specificity; Structural Protein; Technology; Temperature; Testing; Time; Translations; Viral; Visualization; Work; World Health Organization; bacterial genetics; beta-Galactosidase; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; commercialization; cost; detection limit; expectation; improved; lateral flow assay; nanobiosensor; protein expression; rapid detection; recombinase; resistance gene; sensor; synthetic biology; user-friendly; water testing

Summary Phages are the natural viral predators of bacteria and are harmless to humans. The phages’ ability to recognize, bind, infect, and lyse host bacteria has led to their use as sensors for their hosts. Phages can be genetically engineered to express reporter proteins during the infection of their host, resulting in the release of the reporters following the lysis stage of infection. Our central hypothesis is that advanced methods in synthetic biology and genetic engineering will allow phages to be genetically optimized to perform as a dedicated nanobiosensor, and that phage-based assays can deliver genetic-level specificity of their host through reporter selection and optimization. The objective of this proposal is to use synthetic biology to abate current weaknesses identified for phage-based biosensing. The rationale for the proposed research is that while several weaknesses have been proposed for phage biosensors, advances in bioengineering and synthetic biology can provide solutions to address them. These issues include a lack of specificity within a species and a need for increased sensitivity. By mitigating these issues, phage-based sensors can be used to sensitively detect bacterial genetic sequences (e.g., pathogenicity and antibiotic resistance genes) with rapid results, low-cost, and minimal reagent storage conditions. Aim 1: Bioengineering phage infections for maximal reporter protein expression. Our working hypothesis is that the T4 infection conditions can be engineered to maximize the expression of reporter protein by optimizing lysis time and deleting genes non-essential for reporter expression on the phage genome. We will test the working hypothesis by engineering T4 phages with nonessential and structural protein genes deleted/silenced and complemented on a plasmid for propagation. Our expectation is a significant improvement in the limit of detection when used to detect wild type E. coli. Aim 2: Phage-enabled Recombinase Polymerase Amplification (RPA). Our working hypothesis, based on preliminary work, is that T4 phages bioengineered to have genes for RPA proteins can enable the genetic amplification of their host bacteria DNA following phage- induced lysis. To test the working hypothesis, we will genetically engineer the phage T4 with the genes for RPA- required proteins which will be expressed during infection of the bacterial host. Our expectation is that the post- infection lysate mixed with specific primers will allow detection of E. coli with genetic specificity. Following successful completion of the specific aims, the expected outcome is development of a suite of technologies which can bring phage-based biosensing across the technological “valley of death” and towards further application and commercialization. Phages, which allow rapid and low-cost detection of bacteria, suffer from a lack of specificity within a species. We will have demonstrated a method to significantly improve the expression of reporter proteins and a method to provide genetic level specificity while maintaining the overall benefits of phage-based detection (e.g., low-cost, rapid analysis, minimal reagent storage, bacterial lysis).

总结 噬菌体是细菌的天然病毒捕食者，对人类无害。它的识别能力， 结合、感染和裂解宿主细菌导致它们被用作宿主的传感器。噬菌体可以在基因上 被工程化以在其宿主感染期间表达报告蛋白，导致报告蛋白的释放 在感染的溶解阶段之后。我们的中心假设是，先进的合成生物学方法和 基因工程将允许生物传感器进行基因优化，以作为专用的纳米生物传感器， 基于噬菌体的测定可以通过报告基因选择传递其宿主的遗传水平特异性， 优化.该提案的目的是利用合成生物学来消除目前已发现的弱点 用于噬菌体生物传感。拟议研究的理由是，虽然有几个弱点， 被提议用于噬菌体生物传感器，生物工程和合成生物学的进展可以提供解决方案， 解决他们。这些问题包括物种内缺乏特异性以及需要增加灵敏度。通过 为了缓解这些问题，基于噬菌体的传感器可以用于灵敏地检测细菌基因序列， (e.g.,致病性和抗生素抗性基因），结果快速、成本低、试剂储存量少 条件目的1：利用生物工程噬菌体感染实现报告蛋白的最大表达。我们的工作 一种假设是T4感染条件可以被工程化以使报告蛋白的表达最大化 通过优化裂解时间和删除噬菌体基因组上报告基因表达非必需的基因。我们将 通过用非必需和结构蛋白基因改造T4 β来检验工作假设 缺失/沉默并在质粒上互补用于繁殖。我们的期望是一个重大的改进 当用于检测野生型E时，杆菌目的2：噬菌体激活的噬菌体蛋白酶聚合酶 扩增（RPA）。我们的工作假设，基于初步工作，是T4的生物工程， 具有RPA蛋白基因的细菌可以使其宿主细菌DNA在噬菌体- 诱导裂解。为了验证工作假设，我们将用RPA基因对噬菌体T4进行遗传工程改造- 所需的蛋白质将在细菌宿主感染期间表达。我们的期望是，后- 感染裂解物与特异性引物混合将允许检测E.大肠杆菌具有遗传特异性。以下 成功完成具体目标，预期成果是开发一套技术 这可以使基于噬菌体的生物传感技术跨越技术的“死亡谷”，并进一步 应用和商业化。噬菌体，允许快速和低成本的细菌检测，遭受 在一个物种中缺乏特异性。我们将展示一种方法，以显着提高表达 以及提供遗传水平特异性同时保持以下总体益处的方法： 基于噬菌体的检测（例如，低成本、快速分析、最小试剂储存、细菌裂解）。