CAREER: Harnessing Endogenous Defense Systems as Genetic Tools for Microbial Communities

这个CAREER项目由MCB的系统与合成生物学项目和CBET的生物技术与生化工程项目资助，目标是使非模式微生物的遗传工具开发成为一个简单而直接的过程。预计的方法是利用共同的防御系统作为开发不同微生物遗传工具的基础。虽然防御系统自然排斥外来DNA，但这些系统有可能被利用为有效引入重组DNA和执行遗传操作的标准工具。如果成功，由此产生的工具可能会彻底改变我们研究和设计微生物群落成员的能力，进而影响从改善作物健康到防治害虫的各个领域。与拟议的研究相结合，该项目将开发一门新的基因组工程实验室课程，并为罗利东南部的当地社区中心提供科学和工程活动，以吸引周围社区的贫困青年。技术：本研究的长期目标是开发微生物遗传工具开发的标准化方法。这个研究项目解决了两个最艰巨的挑战：有效地转化重组DNA和操纵基因序列和表达。工作假设是，微生物的限制性修饰系统和CRISPR-Cas系统可以用来克服这两个挑战。为了探索这一假设，该项目将研究如何在大肠杆菌中重建宿主细菌的甲基化模式，从而绕过宿主的限制性修饰系统。此外，该项目将研究宿主的原生CRISPR-Cas系统在多大程度上可以用于基因组编辑和转录调控。人类肠道微生物群（一个数据丰富的群落）共有的三种微生物将被调查。其中两种微生物，直肠真杆菌和体coprevotella，来自丰富的系统发育类群，缺乏任何遗传工具，而第三种微生物，大肠杆菌，具有广泛的工具，因此可以作为计划工作的有用起点。

The goal of this CAREER project, funded by the Systems and Synthetic Biology Program in MCB and the Biotechnology and Biochemical Engineering Program in CBET, is to make genetic tool development in non-model microorganisms a simple and straightforward process. The projected approach is to harness common defense systems as the basis for developing genetic tools for diverse microbes. While defense systems naturally reject foreign DNA, these systems have the potential to be harnessed as standard tools to efficiently introduce recombinant DNA and to perform genetic manipulations. If successful, the resulting tools could revolutionize our ability to study and engineer members of microbial communities, in turn impacting areas ranging from improving crop health to combatting insect pests. In conjunction with the proposed research, the project will develop a new laboratory course on genome engineering as well as equipping local community centers in Southeast Raleigh with science and engineering activities to engage underprivileged youth in the surrounding neighborhoods.Technical: The long-term goal of this research is to develop standardized approaches for genetic tool development in microorganisms. This research project tackles two of the most formidable challenges: efficiently transforming recombinant DNA and manipulating gene sequence and expression. The working hypothesis is that the restriction-modification systems and CRISPR-Cas systems native to a microorganism can be harnessed to overcome both challenges. To explore this hypothesis, the project will investigate how recreating the methylation pattern of a host bacterium in Escherichia coli can bypass the host's restriction-modification systems. In addition, the project will investigate the extent to which the host's native CRISPR-Cas systems can be employed for genome editing and transcriptional regulation. Three microorganisms common to the human gut microbiota, a data-rich community, will be investigated. Two of these microorganisms, Eubacterium rectale and Prevotella stercorea, come from abundant phylogenetic groups lacking any genetic tools, whereas the third, Escherichia coli, has extensive tools and thus serves as a useful starting point for the projected work.