Team:Gothenburg/Parts/Part Submission

Part Submisson

Since our whole generation counter is based on cell cycle specific signals for activation of genes and degradation of proteins, we thought it would be beneficial to add one of the specific degradation signals for yeast into the registry. S. cerevisiae was established as one of the most researched on and industrially used organisms. For this reason, we planned to submit the degradation-tag we fused to the fluorescence proteins to make sure they are degraded and do not leak into the next cycle.

Figure 1. Picture of the part in SnapGene.

This degradation-tag, in short D-tag, is a short part of the cyclin Clb3. It is the sequence for the amino acids of the N-terminal until and including a destruction box, a nine amino acid recognition site with the sequence R-x-x-L-x-x-x-x-N. This sequence is necessary but not sufficient for a specific ubiqitin-mediated degradation and to be found in most of the B-cyclins of yeast [1].
Cyclins bind to cell cycle specific kinases and mediate thereby the progress of the different steps of the cell cycle. The six B-cyclins in yeast (Clb1-6) are responsible for the transition into S- and M-Phase of the cell cycle [2]. With increasing digit the degradation occours earlier in the cell cycle [3]. As we reviewed the sequences of the cyclins it became obvious that the distance of the destruction box to the N-terminal of the protein increases with the digit, stating an inverse relation [4].
To have a suitable time point of degradation for our counter we decided on using the sequence of Clb3.

Summary

Our part submission is a 165 bp long N-terminal sequence of the S.cerevisiae protein Clb3. It is a cell cycle dependend degradation signal for proteins. In the registry you can find our part in the entry BBa_K1503000.

References

1. Zachariae W., Destruction with a box: substrate recognition by the anaphase-promoting complex. Mol Cell. 2004 Jan 16;13(1):2-3.

2. Fitch I, et al. (1992) Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. Mol Biol Cell 3(7):805-18.

3. Arellano M, Moreno S., Regulation of CDK/cyclin complexes during the cell cycle. Int J Biochem Cell Biol. 1997 Apr;29(4):559-73.

4. Sequences of the Clb proteins to be found in the Yeast Genome Database