Template:Nevada/TheAuxinSystem

TIR1 is the E3 ubiquitin ligase associated with the Auxin pathway. It is currently the best known F-box protein for integration into yeast, as other F-box proteins from Arabidopsis have acclimated to less thermally variable environments and have low efficiency and expression in yeast cells. TIR1, when loaded with auxin, will bind more efficiently to its substrate, our protein of interest, and will attract ubiquitin to this complex as a signal for degradation. Once ubiquitinated, this whole complex is sent to the 26s proteasome for digestion. We modified TIR1 with a Myc tag for verification once integrated into yeast cells.

Amplified and tagged TIR1 was then engineered to join a promoter, pTEF, and a reporter, GFP. This whole insert was attached to a bacterial vector and transformed with NEB 10 beta competent cells onto LB-ampicillin agar plates in the preliminary stages. After successful colony growth, we extracted this DNA, purified it, and transformed into yeast cells. Initially we had used an ADH1 promoter but kept producing errors. We switched to a promoter known to work not only with GFP but also with yeast, since that was our final destination.

After miniprep of DNA, the newly engineered vector was linearized and transformed into W303 yeast cells. It was screened via yeast colony PCR.

Green fluorescent protein was extracted from yeast via alkali treatment and analyzed on a Western blot and Coomassie blue assay. Our positive control included GFP from bacteria. We wanted to know that GFP was indeed successfully integrated into W303 yeast cells before our next step, adding auxin. After confirmation of GFP, and after the addition of auxin, we would expect to not see GFP on a Western blot since our whole project is based on its degradation.

A degron tag is part of the SCF degradation pathway. It recognizes and binds AUX/IAA proteins and works as a signal for degradation in conjunction with the assembled complex it encounters (TIR1, auxin, GFP). We chose IAA17 (indole acetic acid 17) as our degron because, like TIR1, it is most thermostable for integration into yeast. IAA17 and pTEF were PCR amplified and verified through gel electrophoresis

We designed primers to isolate pTEF-GFP from the plasmid UCSF sent us, pSV606, and verified through gel electrophoresis. This process is much quicker than performing a restriction digest and purifying our segment of interest.

pTEF-GFP and the IAA17 degron were cloned and transformed into a 2μ plasmid. If successful, we would integrate this plasmid with the engineered one above from W303 and perform experiments with varying concentrations of auxin.

Once the two plasmids just mentioned were joined, auxin was introduced in serial dilutions and the degradation of GFP was monitored using a spectrophotometer as well as Western Blot densitometry.