COI1 is the E3 ubiquitin ligase for the JAZ degron pathway. This is analogous to the TIR1 enzyme in the auxin pathway, but instead it binds coronatine. Normally, COI1 binds JA-Ile (jasmonic acid-isoleucine) but this compound is difficult and expensive to synthesize and cannot be purchased. Coronatine is structurally similar and is manufactured, which is why we used it instead. COI1 was amplified and verified through gel electrophoresis.
Template:Nevada/TheCoronatineSystem
From 2014.igem.org
Synthesis and Engineering of the Coronatine Pathway Components
Just like we tagged TIR1 with Myc, here we tagged COI1 with an HA tag for screening. Amplified and tagged COI1 was then joined to pTEF-GFP, inserted into a bacterial vector and transformed with NEB 10 beta competent cells with LB-ampicillin selection. You may have noticed that we use GFP in both of our systems, though we said our goal was a bioorthogonal system to degrade two proteins simultaneously. At the beginning, our use of RFP was not very successful, so for the interest of making sure we can build a construct that works, we used what we had which was GFP and will later swap it out for mCherry when we juxtapose the systems into yeast.
After the above construct grew successful colonies, we isolated and purified the newly engineered vector and transformed into W303 yeast cells. It was screened via yeast colony PCR.
Protein was extracted from yeast via alkali treatment and analyzed on a Western blot and Coomassie blue assay.