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

这个CAREER项目的目标，由MCB的系统和合成生物学计划以及CBET的生物技术和生物化学工程计划资助，是使非模型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。虽然防御系统天然地拒绝外来DNA，但这些系统具有被利用作为标准工具以有效地引入重组DNA和进行遗传操作的潜力。如果成功，所产生的工具可以彻底改变我们研究和设计微生物群落成员的能力，从而影响从改善作物健康到防治害虫的各个领域。该项目还将开发一个新的基因组工程实验室课程，并为东南部罗利的社区中心提供科学和工程活动，以吸引周边社区的贫困青年。技术：该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。该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，可以利用微生物天然的限制修饰系统和CRISPR-Cas系统来克服这两个挑战。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修饰系统。此外，该项目还将研究宿主的原生CRISPR-Cas系统可用于基因组编辑和转录调控的程度。将研究人类肠道微生物群中常见的三种微生物，这是一个数据丰富的社区。其中两种微生物，直肠真杆菌和粪普雷沃菌，来自丰富的系统发育组，缺乏任何遗传工具，而第三种，大肠杆菌，具有广泛的工具，因此作为一个有用的起点，预计的工作。

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.