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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.
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. .
JAZ1 has already been proven to be a successful degron tag in yeast. We are using this as a positive control while also researching the efficiency of JAZ6 as a degron in yeast. The degron tags were PCR amplified and verified through gel electrophoresis. The UCSF iGEM team also provided a pSV606 plasmid containing the pTEF constitutive promoter and GFP. Through designed primers, we again amplified only the pTEF and GFP out of the plasmid in the interest of time.
pTEF-GFP and the JAZ degrons were cloned and transformed into a 2μ plasmid. Like our auxin pathway, this would then be introduced to the COI1 plasmid and we would continue with our experiments of adding varying concentrations of coronatine.
After the introduction of coronatine, the degradation of GFP was monitored using a spectrophotometer as well as Western Blot densitometry. Because coronatine is to induce the destruction of GFP, we would expect to not see GFP on the Western blot and to only see a band on our positive, bacterial control.
Jaz1 is a degron tag utilized in the coronatine pathway. The protein of interest is tagged with Jaz1; when coronatine is added to the system, the protein and its tag are linked to the E3 ubiquitin ligase, COI1. Click the picture or the name below to be taken to the parts registry page.
RFP-Jaz1 is a degron tag utilized in the coronatine pathway. The protein of interest is tagged with RFP-Jaz1; when coronatine is added to the system, the protein and its tag are linked to the E3 ubiquitin ligase, COI1. Click the picture or the name below to be taken to the parts registry page.
Jaz6 is a degron tag utilized in the coronatine pathway. The protein of interest is tagged with Jaz6; when coronatine is added to the system, the protein and its tag are linked to the E3 ubiquitin ligase, COI1. Click the picture or the name below to be taken to the parts registry page.
COI-1 or Coronatine Insensitive-1 recruits JAZ-1 or JAZ-6 tagged substrates in the presence of Coronatine for degradation by the Proteasome.
TiRI is an ubuquitinase E3 that recognizes AID-tagged proteins and trigger their ubiquitination for further degradation. This part works with K812011 which is a AID tagged GFP. OsTirI is a protein comming from rice that have been identified as working in mammalian cells and yeast. This part takes place in a device pattented by Kanemaki Masato, Kakimoto Tatsuo, Nishimura Kohei, Takisawa Haruhiko and Fukagawa Tatsuo for yeast and mammalian cells use . However the patent does not cover the use for oviparian such as frogs and chicken.
This part is a GFP coding sequence fused to an AID tag for its recognition by the ubiquitinase E3 that induces its degradation in the presence of auxin in the eukaryote cell. This is designed to be used in pSB1C3 vector. This part is a part of a device patented by Kanemaki Masato, Kakimoto Tatsuo, Nishimura Kohei, Takisawa Haruhiko and Fukagawa Tatsuo for yeast and mammalian cells use.
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