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.
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.
