Difference between revisions of "Part:BBa K3506030"

Revision as of 17:18, 21 October 2020

The cassette of periodic expression and degradation of Cas9

This part consists of four parts: CLB2 promoter, the first 124 amino acids of Clb2, a linker and Cas9. CLB2 promoter is a cell-cycle associated promoter and makes Cas9 expressed in the S phase and reached its peak in the G2/M phase. The first 124 amino acids of Clb2 contains D-box and KEN-box, which are the recognition site by E3 ubiquitin ligase APC. The APC mediates the ubiquitin-proteasome proteolysis, making Cas9 degraded quickly. By this way, we control the expression and degradation of Cas9 periodically.

Usage

This part can be used to control Cas9 expression level with yeast cell cycle. If you want to control the expression and degradation of Cas9 during cell division, you can place this part on the yeast plasmid.

Sequence and Features

Design and Properties

We design a module to verify the periodic expression and degradation of Cas9,in which Cas9(BBa_K2130013) linkeded with the first 124 amino acids of Clb2(BBa_K3506000) is put under the CLB2 promoter(BBa_K3506007).

Experimental approach

1. Construct recombinant plasmid. Get Cas9 from the plasmid pRH003.Get CLB2 promoter and the first 124 amino acids of Clb2 from S.cerevisiae S288C by PCR. Ligate the fragments by in-fusion cloning.

2. Transform the product (2.5μL) into DH5α competent cells（50μL）, coat cells on each agar plate (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones were selected for colony PCR. Expanding culture colonies at 37℃ 200rpm,extract plasmids and sequence.

3. Linearize the plasmids with Xho1 and transform them(5-10ng) into S. cerevisiae BY4741. Coat cells on SD-ura plate and incubate at 30℃ for 3 days. Monoclones were selected for colony PCR and sequencing.

4. Synchronize S. cerevisiae cells. Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize yeast cells.

5. Release the yeast cells from synchronization. Remove a time-zero fraction. Collect fractions of culture every 10 min for 120–180 min for Western Blot. Strain without plasmid transformation was used as negative control. Don’t forget to select the internal reference.

6. Obtain and analyze data. Draw the image of Cas9 protein levels over time.

References

[1]Trcek, T. , Larson, D. , Alberto Moldón, Query, C. , & Singer, R. . (2011). Single-molecule mrna decay measurements reveal promoter- regulated mrna stability in yeast. Cell, 147(7), 1484-1497.

[2]Michael D. Mendenhall, & Amy E. Hodge. (1998). Regulation of cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast saccharomyces cerevisiae.Microbiol Mol Biol Rev, 62(4), 1191-1243.

[3]Wu, X., Liu, L., & Huang, M. (2011). Analysis of changes in protein level and subcellular localization during cell cycle progression using the budding yeast Saccharomyces cerevisiae. Methods in molecular biology(Clifton,N.J.),782,47–57.

[4]Manukyan, A. , Abraham, L. , Dungrawala, H. , & Schneider, B. L. . (2011). Synchronization of yeast. Methods in Molecular Biology, 761(761), 173.

[5]Wang, Y. et al. A ‘suicide’ CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans. Sci. Rep. 6, 31145; doi: 10.1038/srep31145 (2016).

[6]Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science (New York, N.Y.), 339(6121), 819–823.

[7]Hendrickson, C., Meyn, M. A., 3rd, Morabito, L., & Holloway, S. L. (2001). The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition. Current biology : CB, 11(22), 1781–1787.