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

基因工程是发现细菌生理学基本方面的有力方法， 代谢和发病机制。问题是绝大多数可以在实验室中培养的细菌 在遗传学上仍然是棘手的，超出了遗传学的力量，无法阐明功能或为人类工程 使用.遗传学的难题阻碍了基础、合成和预防微生物学的研究 发展先行者的要求研究人员花了数年时间一个物种一个物种地构建特定的遗传系统， 艰苦和昂贵的过程。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遗传上易于处理，而不管其分类谱系或遗传和 物理屏障。我们希望我们的方法将改变医学，环境和生物学领域的微生物研究。 生物技术.我们的SyngenicDNA-μPOET（基于电穿孔的微流控参数优化 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 技术，合作设计，以克服两个根本原因的遗传棘手的大多数 细菌第一项新技术SyngenicDNA克服了复杂的细菌防御机制， 通过使用快速宿主模拟策略降解非自身DNA。这种新的策略重新编码了任何 遗传工具（例如，质粒或转座子）以消除由特异性抗体识别的靶非自身标记。 目的细菌菌株，从而防止通过先天限制性修饰（RM）的DNA降解， 规则间隔短回文重复序列（CRISPR）-Cas系统。第二个新 μPOET技术使用微流体和机器人技术克服了非自身DNA进入的物理障碍。 μPOET利用高通量微流控电穿孔技术创建一个兼容 使用96孔板液体处理系统，可以快速筛选电穿孔条件， 比传统的基于比色皿的方法快几个数量级。一旦建立，SyngenicDNA- μPOET平台将成为一种资源，允许在几乎任何可培养的植物中产生遗传易处理性。 细菌种类在几周内，而不是几年。作为原则的证明，我们将展示 SyngenicDNA-μPOET平台在人类口腔微生物组上的应用。缺乏遗传上易驾驭的 细菌是破译人类微生物组成员的功能属性的巨大挑战。 我们将扩展现有的人类口腔微生物组数据库（HOMD），并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种模型菌株的初始储存库 在口腔微生物组内的六个不同的门中，每个门都使用SyngenicDNA进行遗传处理- μPOET平台。这一资源将迅速加速对口腔物种在生物多样性中的作用的基础研究。 人类健康和疾病。我们的首要目标是提供一种普遍适用的方法， 使大多数细菌在遗传上易于控制。

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.