The SyngenicDNA and μPOET Platform: Overcoming Innate Barriers to Genetic Engineering in Bacteria.

SyngenicDNA 和 μPOET 平台：克服细菌基因工程的先天障碍。

• 批准号: 9369398
• 负责人: Christopher D Johnston
• 金额: $ 156.51万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-08 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9369398
• 关键词: Bacteria; Bacterial Model; Bacterial Physiology; Biotechnology; Bypass; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Common Core; Communities; Complex; Computer Simulation; DNA; DNA Methylation; Databases; Defense Mechanisms; Development; Disease; Ecosystem; Electroporation; Environment; Epigenetic Process; Equipment; Generations; Genetic; Genetic Engineering; Genetic Template; Goals; Habitats; Health; Human; Human Engineering; Human Microbiome; Income; Individual; Industrialization; Institutes; Investigation; Laboratories; Liquid substance; Medicine; Metabolism; Methodology; Methods; Methylation; Microbe; Microbiology; Microfluidics; Modification; Oral; Pathogenesis; Plasmids; Process; Research; Research Personnel; Resources; Robotics; Role; Single Nucleotide Polymorphism; Site; Skin; System; Taxonomy; Technology; Time; United States National Institutes of Health; bacterial genetics; base; design; field study; innovation; instrument; interest; member; microbial; microbiome; new technology; next generation sequencing; novel; novel strategies; operation; oral microbiome; prevent; repository; research and development; screening; synthetic construct; tool; virtual

Genetic engineering is a powerful approach for discovering fundamental aspects of bacterial physiology, metabolism, and pathogenesis. The problem is the vast majority of bacteria that can be grown in a laboratory remain genetically intractable, beyond the power of genetics for elucidating function or for engineering for human use. The challenge of genetic intractability stymies basic-, synthetic-, and translational-microbiology research and development. Researchers spend years constructing ad hoc genetic systems one species at a time, an arduous and expensive process. Here, we introduce a groundbreaking, rapid, broadly applicable technology for rendering any cultivable bacterial species genetically tractable, irrespective of taxonomic lineage or genetic and physical barriers. We expect our approach will transform microbial research in medicine, the environment, and biotechnology. Our SyngenicDNA-μPOET (Microfluidic Parametric Optimization of Electroporation based Transformation) platform is a combination of two entirely novel, broadly applicable, and currently unavailable technologies, co-operatively designed to overcome the two underlying causes of genetic intractability within most bacteria. The first new technology, SyngenicDNA, overcomes the complex bacterial defense mechanisms that degrade non-self DNA by using a rapid host-mimicking strategy. This novel strategy recodes the DNA of any genetic tool (e.g., plasmids or transposons) to eliminate target non-self signatures recognized by a specific bacterial strain of interest, thus preventing DNA degradation by innate Restriction Modification (RM) and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems. The second new technology, μPOET, overcomes the physical barrier to non-self DNA entry using microfluidics and robotics. μPOET leverages high-throughput microfluidic electroporation to create a transformation platform compatible with 96-well plate liquid handling systems to enable rapid screening of electroporation conditions, two to three orders of magnitude faster than traditional cuvette based approaches. Once established, the SyngenicDNA- μPOET platform will be a resource allowing the generation of genetic tractability in virtually any cultivable bacterial species over the span of weeks, rather than years. As proof of principle, we will demonstrate the power of the SyngenicDNA-μPOET platform on the human oral microbiome. The paucity of genetically tractable bacteria is a formidable challenge to deciphering the functional attributes of members of the human microbiome. We will expand the current Human Oral Microbiome Database (HOMD) and establish the Human Oral Microbiome Culture (HOMC) collection: an initial repository of 200 model bacterial strains representing species across six different phyla within the oral microbiome, each made genetically tractable using the SyngenicDNA- μPOET platform. This resource will rapidly accelerate fundamental investigations into the role of oral species in human health and disease. Our overarching goal is to provide a universally applicable methodology to rapidly render most bacteria genetically tractable.

基因工程是一种发现细菌生理基本方面的有力方法， 代谢和发病机理。问题是可以在实验室中生长的绝大多数细菌 在遗传上仍然棘手，超出了阐明功能的遗传学能力或人类工程 使用。遗传性棘手性的挑战Stymies基本，合成和翻译 - 微生物学研究 和发展。研究人员花费数年的时间来构建一个临时遗传系统一次，一个物种，一个 艰巨而昂贵的过程。在这里，我们介绍了一项开创性，快速，广泛适用的技术 呈现任何可栽培的细菌在遗传上可造成的物种，无论分类学谱系或遗传性如何 身体障碍。我们预计我们的方法将改变医学，环境和环境的微生物研究 生物技术。我们的SyngenicDNA-μpoet（基于电穿孔的微流体参数优化 转换）平台是两个完全新颖的，广泛适用且目前不可用的组合 合作设计的技术，旨在克服大多数遗传性疾病的基本原因 细菌。第一个新技术SyngenicDNA克服了复杂的细菌防御机制 通过使用快速宿主模仿策略来降低非自我DNA。这种新颖的策略重新编码了任何的DNA 遗传工具（例如质粒或转座子）消除了特定的目标非自动特征 细菌感兴趣的菌株，从而防止了通过先天限制修饰（RM）和 群集定期间隔短的短质体重复（CRISPR）-CAS系统。第二个新 技术，μpoet，使用微富集剂和机器人技术克服了非自动DNA进入的物理障碍。 μpoet利用高通量微流体电穿孔来创建一个兼容的转换平台 使用96孔板液体处理系统，可以快速筛选电穿孔条件，两到三 比基于传统比色杯的方法快的数量级。建立后，SyngenicDNA- μpoet平台将是一种资源，允许几乎任何可耕种的遗传障碍性生成 在几周而不是几年的跨越细菌物种。作为原则的证明，我们将展示力量 人口腔微生物组上的Syngenicdna-μpoet平台的。一般拖延的匮乏 细菌是破译人类微生物组成员的功能属性的巨大挑战。 我们将扩展当前的人类口服微生物组数据库（HOMD）并建立人类口头 微生物组培养（HOMC）集合：代表物种的200个模型细菌菌株的初始存储库 在口服微生物组内的六个不同的门上，每个门都可以使用SyngenicDNA-逐渐拖动 μpoet平台。该资源将迅速将基本投资加速到口头物种中的作用 人类健康和疾病。我们的总体目标是提供一种普遍适用的方法来快速 渲染大多数可促成的细菌。