Difference between revisions of "Part:BBa K2019003"

Latest revision as of 23:19, 19 October 2016

Periplasm Directed saCas9 via TorA Signaling Sequence (TorA-saCas9)

TorA-Cas9 is a fusion of the TorA signaling sequence to the N-terminus of staphylococcus aureus CRISPR associated protein 9 (saCas9). TorA utilizes the twin arginine translocation (tat) pathway to direct the fused protein to the periplasm of the cell. Therefore, by fusing Cas9 to the C-terminus of the signaling sequence, it should be directed to the periplasm of the cell.

Usage and Biology

Usage and Biology

We tested to see if saCas9 was properly directed to the periplasm of the cell. First, we used the cold osmotic shock protocol from the 2014 Bielefeld iGEM team to create a periplasm protein fraction of BBa_K2019003 being expressed in the BBa_R0040 backbone in a top10 E. Coli strain. Furthermore, we also utilized an OMV purification protocol by the 2016 UNSW iGEM team to see if Cas9 was being incorporated in outer membrane vesicles. To do this, we transformed our BBa_K2019003-BBa_R0040 expression system into a hypervesiculating E. Coli cell line JC8031 and then ran the OMV purification protocol on the product. Our results displayed that there was no TorA-Cas9 expressed in the periplasm of the cell. However, there were several possible issues with the procedure for the expression test of TorA-Cas9; thus, we are unsure if this part successfully translocates Cas9 to the periplasm.

Sequence and Features