THE SYNGENICDNA AND UPOET PLATFORM: OVERCOMING INNATE BARRIERS TO GENETIC ENGINEE

SYNGENICDNA 和 UPOET 平台：克服遗传引擎的先天障碍

• 批准号: 10632208
• 负责人: Christopher D Johnston
• 金额: $ 64.16万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10632208
• 关键词: SYNGENICDNA; UPOET; PLATFORM; OVERCOMING; INNATE

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，

项目成果

The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota.

• DOI: 10.1016/j.celrep.2022.111625
• 发表时间: 2022-11-15
• 期刊: Cell reports
• 影响因子: 8.8

Complete Genome Sequence of Clostridium cadaveris IFB3C5, Isolated from a Human Colonic Adenocarcinoma.

• DOI: 10.1128/mra.01135-21
• 发表时间: 2022-03-17
• 期刊: Microbiology resource announcements
• 影响因子: 0.8
• 作者: McGlinchey AS;Zepeda-Rivera MA;Stepanovica M;Baryiames AA;Jones DS;LaCourse KD;Bullman S;Johnston CD
• 通讯作者: Johnston CD

Corrigendum to "Expanding the rumen Prevotella collection: the description of Prevotella communis, sp. nov. of ovine origin" [Syst. Appl. Microbiol. 46(4) (2023) 126437].

“扩大瘤胃普雷沃氏菌收集：绵羊起源普雷沃氏菌，sp. nov. 的描述”的勘误表 [Syst.

• DOI: 10.1016/j.syapm.2023.126453
• 发表时间: 2023
• 期刊: Systematic and applied microbiology
• 影响因子: 3.4
• 作者: Grabner,Eva;Stare,Eva;Fanedl,Lijana;Zorec,Maša;Jones,DakotaS;Johnston,ChristopherD;Avguštin,Gorazd;Accetto,Tomaž
• 通讯作者: Accetto,Tomaž

Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators.

• DOI: 10.1038/s41467-018-05525-6
• 发表时间: 2018-08-06
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Juárez JF;Lecube-Azpeitia B;Brown SL;Johnston CD;Church GM
• 通讯作者: Church GM

INVADEseq to identify cell-adherent or invasive bacteria and the associated host transcriptome at single-cell-level resolution.

• DOI: 10.1038/s41596-023-00888-7
• 发表时间: 2023-11
• 期刊: NATURE PROTOCOLS
• 影响因子: 14.8
• 作者: Nino, Jorge Luis Galeano;Wu, Hanrui;Lacourse, Kaitlyn D.;Srinivasan, Harini;Fitzgibbon, Matthew;Minot, Samuel S.;Sather, Cassie;Johnston, Christopher D.;Bullman, Susan
• 通讯作者: Bullman, Susan