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

这一职业项目由MCB的系统和合成生物学计划以及CBET的生物技术和生化工程计划资助，其目标是使非模式微生物中的遗传工具开发成为一个简单和直接的过程。预计的方法是利用共同的防御系统作为开发针对不同微生物的遗传工具的基础。虽然防御系统自然会拒绝外来DNA，但这些系统有可能被用作标准工具，有效地引入重组DNA并进行遗传操作。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，进而影响到从改善作物健康到抗击虫害的各个领域。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配备科学和工程活动，以吸引周围社区的贫困青年。技术：这项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。这项研究项目解决了两个最艰巨的挑战：高效转化重组DNA和操纵基因序列和表达。工作假设是，微生物固有的限制性内切酶修饰系统和CRISPR-Cas系统可以被利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，以绕过宿主的限制修饰系统。此外，该项目还将调查寄主的本地CRISPR-CAS系统可用于基因组编辑和转录调控的程度。将调查人类肠道微生物区系的三种常见微生物，这是一个数据丰富的群落。其中两种微生物，直肠真核细菌和斯特氏普雷沃特氏菌，来自缺乏任何遗传工具的丰富的系统发育群，而第三种微生物，大肠杆菌，拥有广泛的工具，因此是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.