Team:Northwestern/Design
Project design
This summer, our team tackled three key features of this project: Cas9 expression, its packaging into OMVs and finally the fusion of these vesicles with target cells. To study these three areas we made use of a E.coli strain that is genetically engineered to hypervesiculate (JC8031). These bacteria were transformed with a plasmid that allows the metabolic production of the Cas9 protein attached to amino acid sequences which were identified as good candidates for periplasmic localization. To test for the presence of Cas9 in different cell compartments we made use of fractionation - lysing only the outer membrane of the cells.
Translocation pathways
Fusion proteins were employed for Cas9 export to to the periplasm of E.coli cells. We aimed to examine the possibility of exporting a large protein, saCas9, through the Twin arginine translocation pathway (Tat), general secretory pathway (Sec) or both. Proteins that are able to fold in their 3D conformation in the cell cytoplasm ....
