Part:BBa_K2019003
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. Results of the two experiments can be seen in the western blot figures below:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 753
Illegal BglII site found at 906
Illegal BglII site found at 1052 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 326
Illegal AgeI site found at 2882 - 1000COMPATIBLE WITH RFC[1000]
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