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.

基因工程是发现细菌生理学基本方面的有力途径， 代谢，以及发病机制。问题是绝大多数细菌都可以在实验室里培养 保持遗传上的顽固性，超出了遗传学解释功能或人类工程学的能力 使用。基因难解性的挑战阻碍了基础微生物学、合成微生物学和翻译微生物学研究 和发展。研究人员花费数年时间一次构建一个物种的特殊遗传系统， 这一过程费时费力。在这里，我们介绍一种突破性的、快速的、广泛适用的技术 使任何可培养的细菌物种在基因上易于处理，而无论分类谱系或遗传和 身体上的障碍。我们希望我们的方法将改变医学、环境和环境领域的微生物研究 生物技术。我们的SyngenicDNA-μPET(基于微流控参数优化的电穿孔) 转换)平台是两个全新的、广泛适用的、目前不可用的平台的组合 合作设计的技术，以克服大多数人的遗传难题的两个根本原因 细菌。第一项新技术SyngenicDNA克服了复杂的细菌防御机制 使用一种快速模仿宿主的策略来降解异体DNA。这一新的策略重新编码了任何 一种基因工具(例如，质粒或转座子)，用于消除特定基因识别的目标非我特征 感兴趣的细菌菌株，从而通过先天性限制性内切酶修饰(RM)和 簇状规则间隔短回文重复(CRISPR)-CAS系统。第二条新消息 技术，μ诗人，克服了使用微流体和机器人进入非我dna的物理障碍。 μPOTE利用高通量微流控电穿孔创建兼容的转换平台 配备96孔板液体处理系统，能够快速筛选电穿孔条件，两到三个 比传统的试管方法快几个数量级。一旦建立起来，合成DNA- μPEET平台将成为一种资源，允许在几乎任何可栽培的作物中产生遗传适应性 细菌种类在几周内，而不是几年内。作为原则性的证明，我们将证明 人类口腔微生物组上的SyngenicDNA-μ诗人平台。遗传上易驯服的基因的缺乏 细菌对破译人类微生物组成员的功能属性是一个巨大的挑战。 我们将扩展现有的人类口腔微生物组数据库(HOMD)，建立人类口腔微生物组数据库 微生物组培养(HOMC)收集：代表物种的200个模式细菌菌株的初始储存库 在口腔微生物群中的六个不同的门中，每个都使用SyngenicDNA使其在遗传上易于处理- μ诗人平台。这一资源将迅速加速对口腔物种在 人类的健康和疾病。我们的首要目标是提供一种普遍适用的方法，以迅速 使大多数细菌在基因上易于驯服。