Bioengineering Phage-based Biosensors with Genetic Specificity and High Sensitivity

摘要 噬菌体是细菌的天然病毒捕食者，对人类无害。噬菌体的识别能力， 结合、感染和裂解宿主细菌导致它们被用作宿主的传感器。噬菌体可以从基因上 被设计为在感染宿主期间表达报告蛋白，导致记者释放 在感染的裂解阶段之后。我们的中心假设是合成生物学中的先进方法和 基因工程将允许噬菌体经过基因优化，作为专用的纳米生物传感器发挥作用，以及 基于噬菌体的检测可以通过报告选择和选择传递宿主的遗传水平的特异性 优化。这项提议的目标是使用合成生物学来消除目前已发现的弱点。 用于基于噬菌体的生物传感。提出这项研究的理由是，尽管有几个弱点 已被提出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 给他们讲讲。这些问题包括物种内缺乏特异性，以及需要提高敏感性。通过 为了缓解这些问题，基于噬菌体的传感器可以用于敏感地检测细菌遗传序列 (例如，致病性和抗生素抗性基因)具有快速、低成本和最少的试剂存储 条件。目的1：生物工程噬菌体感染以获得最大的报告蛋白表达。我们的工作 假设T4感染条件可以被改造为最大限度地表达报告蛋白 通过优化裂解时间和删除噬菌体基因组上对报告基因表达不必要的基因。我们会 用非必需和结构蛋白基因改造T4噬菌体来检验工作假说 缺失/沉默，并在用于繁殖的质粒上进行补充。我们的预期是一个显著的进步 用于检测野生型大肠埃希氏菌时的检出限。目的2：噬菌体激活的重组酶聚合酶 放大(RPA)。我们的工作假设是，基于初步工作，T4噬菌体被生物工程改造成 具有RPA蛋白的基因可以使其宿主细菌DNA在噬菌体- 诱导溶解。为了验证工作假说，我们将用RPA-基因对噬菌体T4进行基因工程。 在细菌宿主感染期间将表达的所需蛋白质。我们的预期是邮局- 将感染裂解物与特定的引物混合，可以检测出具有遗传特异性的大肠杆菌。跟随 圆满完成具体目标，预期成果是开发出一套技术 这将使基于噬菌体的生物传感跨越技术的“死亡之谷”，并进一步 应用和商业化。噬菌体可以快速和低成本地检测细菌，但受到 在一个物种内缺乏专一性。我们将演示一种显著改善表达的方法 以及一种提供遗传水平特异性而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、最小试剂存储、细菌裂解)。

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).