Part:BBa_K3506030

The cassette of periodic expression and degradation of Cas9

This module 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 at S phase and reached its peak at G2/M phase. The first 124 amino acids of Clb2 contains D-box and KEN-box, which are the recognition sites 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 module can be used to control Cas9 expression with yeast cell cycle. If you want to control the expression and degradation of Cas9 during cell division, you can place this part on a yeast plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 229
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1578
  Illegal BglII site found at 2373
  Illegal BamHI site found at 2667
  Illegal XhoI site found at 203
  Illegal XhoI site found at 3173
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2405
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1496
  Illegal BsaI.rc site found at 2738
  Illegal BsaI.rc site found at 4094
  Illegal BsaI.rc site found at 4517
  Illegal BsaI.rc site found at 4529
  Illegal BsaI.rc site found at 5396
  Illegal SapI.rc site found at 4201
  Illegal SapI.rc site found at 4783
  Illegal SapI.rc site found at 4798
  

Design

We design a module to verify the periodic expression and degradation of Cas9, in which Cas9(BBa_K2130013) fused to the first 124 amino acids of Clb2 (BBa_K3506010) is put under the control of the CLB2 promoter(BBa_K3506007).

Experimental approach

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

2. Transform the product (2.5μL) into E.coli DH5α competent cells（50μL）, grow cells on agar plates (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones were screened by colony PCR. The proper colony was selected and cultured at 37℃ and 200rpm, extract plasmids and sequence.

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

4. Synchronize Saccharomyces cerevisiae cells.

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

5. Release the yeast cells from synchronization. Collect a time-zero fraction. Then collect fractions of culture every 10 min for 120–180 min for Western Blot. Strain without being transformed was used as the 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.