Difference between revisions of "Team:Northwestern/experiments"

Packaging Cas9

In order to successfully package Cas9 into Outer Membrane Vesicles, we needed a source of OMVs. We transformed our saCas9 encoding plasmids into a hyper-vesiculating strain of E. coli known as JC8031. This strain is genetically engineered to create a large amount of OMVs and as a result, any substance whose concentration is high in the periplasm of the cell would also be expected to exist within the generated OMVs. The Cas9 itself also has a His6 tag attached for identification purposes, as well as a signal sequence meant to direct and secrete the protein into the periplasm.

Signaling Sequences

The following table includes the signalling sequences that we used and tested over the course of the summer. They are divided into the secretion pathway taken by the sequences, where an ambiguous sequence simply has a less defined pathway or may use multiple pathways.

In order to determine which secretion tags would be of most use to us, and because the tag is expected to detach from the Cas9 protein after successful delivery to the periplasm, we performed Signal Immunoprecipitation (IP) on our signalling sequences. This analysis gave us information regarding where the cleavage of the tag from the protein would most likely occur and what the likelihood is of this event occurring. Below is an example of a Signal IP for PelB which is one of our more useful secretion tags.

In addition to the signalling sequences, we also fused the Cas9 protein to ClyA, a pore-forming toxin, which was expected to bore through the inner membrane of the cell and lodge itself in the outer membrane, eventually having an OMV for around it. This way the Cas9 would follow the ClyA into the OMVs.

Types of Cas9 Protein

Two different types of Cas9 were characterized by our team over the summer. The saCas9 already existed in the iGEM registry having been used by previous teams. However, in order to explore more options and find a more compatible Cas9, we also used cjCas9 protein. This Cas9 has two main benefits to our project. The first is that the size of cjCas9 is comparatively much smaller than the saCas9, making it more likely that this protein will fit inside the JC8031 OMVs. Secondly, the cjCas9 has been demonstrated to have much less off-target effects and thereby target the gene of interest with much more efficiency than saCas9. These two differences make cjCas9 the apparent optimal protein for our project, but further comparative testing of these two proteins give more information of their individual benefits.

Types of Cas9 Protein

One important experiment performed during the course of this project was a Cas9 functionality test to prove that our cas9 was functionally operational inside the JC8031 cells. The protocol of this experiment involved four separate transformations into JC8031 using 3 different plasmids: