Functional Metagenomic Reprogramming of the Human Microbiome through Mobilome Eng

项目摘要 这项研究的长期目标是制定文化独立的战略，以有效地工程 并在体内编程人类微生物组。为此，本提案的目标是制定一项 使能技术平台，命名为基因组工程的自我传输复制（GESTR），允许 外源基因在人类微生物群中的有效递送、繁殖和表达，以表征 微生物组中基因繁殖的动力学，并初步证明这些技术在 改造小鼠的肠道微生物群。这项工作的具体目标是：（一）构建工程化的自我- 传递含有可追踪的有效载荷基因的接合转座子，并表征其 动员到通常在人类肠道微生物群中发现的不同菌株中; ii）开发有效载荷文库 用于表征转录效率和密码子适应以实现最佳有效负载基因表达的构建体 在优势肠道微生物中;和iii）通过工程化的方法测量外源基因的基因流动和传递， 在小鼠模型的肠道微生物群中的可自我动员的遗传元件。新开发的工具包括 多重自动基因组工程，从头DNA微阵列为基础的基因合成，和Meta- 转录组学将被用来促进这个项目的发展。该研究将提供 第一次演示关键功能，并发展足够的新理解， 在未来的应用中更广泛地使用该技术平台。传输动力学中的关键问题 和人类微生物组中横向共享基因的自然选择也将得到解决。工程 人类微生物组报告、预防和逆转疾病状态的能力增强， 调节食物和药物的新陈代谢有望成为改变人类- 相关微生物进入微型传感器，微型蛋白质生产工厂，以及适应性生物修复 系统.该技术具有开发临床治疗常见微生物的潜力- 相关疾病，如肠道疾病（例如克罗恩病、IBD、慢性消化不良）、口腔疾病（例如牙科 龋齿）、泌尿生殖道（例如感染、STD）和皮肤（例如异位湿疹）。

Project Summary The long-term goal of this research is to develop culture-independent strategies to effectively engineer and program the human microbiome in vivo. To this end, the objective of this proposal is to develop an enabling technology platform, named Genome Engineering by Self-Transmissible Replicons (GESTR), to allow the efficient delivery, propagation and expression of exogenous genes in the human microbiota, to characterize the kinetics of gene propagation in the microbiome, and to initially demonstrate the utility of such techniques in engineering the gut microbiota of mice. The specific goals of this work are i) to construct engineered self- transmitting conjugative transposons containing trackable payload genes and characterize their ability to mobilize into different strains typically found in the human gut microbiota; ii) to develop libraries of payload constructs to characterize transcriptional efficiency and codon adaptation for optimal payload gene expression in dominant gut microbes; and iii) to measure gene flow and transmission of exogenous genes by engineered self-mobilizable genetic elements in the gut microbiota of a murine model. Newly developed tools including Multiplex Automated Genome Engineering, de novo DNA-microarray-based gene synthesis, and meta- transcriptomics will be utilized to facilitate the development of this project. The proposed research will provide the first demonstration of key functionality and the development of sufficient new understanding to enable the broader use of this technology platform in future applications. Key questions in the dynamics of transmission and natural selection of laterally shared genes in the human microbiome will also be addressed. Engineering the human microbiome with augmented capabilities to report, prevent and reverse disease states and to modulate the metabolism of foods and drugs promises to be a critical avenue towards transforming human- associated microbes into micro-sensors, miniature protein-production factories, and adaptive bioremediation systems. This technology holds potential for development of clinical therapeutics of common microbial- associated diseases such as those of the gut (e.g. Crohn's, IBD, chronic maldigestion), oral cavity (e.g. dental caries), urogenital tract (e.g. infections, STDs), and skin (e.g. ectopic eczema).