Part:BBa_K2019004
Periplasm Directed saCas9 via DsbA Signaling Sequence (DsbA-saCas9)
DsbA-Cas9 is a fusion of the DsbA signaling sequence to the N-terminus of staphylococcus aureus CRISPR associated protein 9 (saCas9). DsbA is cotranslational and utilizes the SRP pathway to direct fused proteins to the periplasm of the cell.
Usage and Biology
We tested to see if saCas9 was properly directed to the periplasm of the cell. The first method we utilized is the cold osmotic shock protocol from the 2014 Bielefeld iGEM team to create a periplasm protein fraction of BBa_K2019004 being expressed in the BBa_R0040 backbone in a top10 E. Coli strain. Furthermore, we also received an OMV purification protocol from the 2016 UNSW iGEM team and utilized it to see if Cas9 was being incorporated in outer membrane vesicles. To do this, we transformed our BBa_K2019004-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 DsbA-Cas9 expressed in the periplasm of the cell. However, there were several possible issues with the procedure for the expression test of DsbA-Cas9; thus, we are unsure if this part successfully translocates Cas9 to the periplasm.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 783
Illegal BglII site found at 936
Illegal BglII site found at 1082 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 356
Illegal AgeI site found at 2912 - 1000COMPATIBLE WITH RFC[1000]
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