Project Abstract

We focused on establishing a framework to implement genetic manipulation techniques—specifically, multiplex automated genome engineering (MAGE) and CRISPR-Cas9 systems—into non-model, environmentally significant microbes using standard biological parts. The framework involves two components: (1) propagation and selection of cultures and (2) manipulation of cell genomes by MAGE and/or CRISPR. We identified design considerations for both components of the framework, and experimentally validated propagation and selection considerations using cyanobacterial strain Synechococcus sp. PCC 7002 (a fast-growing cyanobacterium capable of lipid biofuel production) and Rhizobium tropici CIAT (a nitrogen-fixing rhizobium which forms root nodules in legume plants). We then developed a workflow for the design, construction, and testing of MAGE and CRISPR technologies into non-model prokaryotes. The insights we gained from validating the propagation component of our workflow will serve to improve the versatility and robustness of our framework and will inform the development of tools for genetic manipulation in other non-model organisms.