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方法，