Difference between revisions of "Part:BBa K3506009"

Latest revision as of 09:18, 27 October 2020

The cassette of periodic expression of Cas9

This part consists of two parts: CLB2 promoter and Cas9. Combining these two parts enables the CLB2 promoter to control the expression of Cas9, so as to achieve the goal of making the expression of Cas9 with the cell cycle. Prospectively, Cas9 expression begins at S phase and reaches its peak at G2/M phase.

Usage

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

Sequence and Features

Design

We design a module to verify the periodic expression of Cas9,in which the gene of Cas9 (BBa_K2130013) is put under the control of CLB2 promoter (K3506007).

Experimental approach

1. Construct recombinant plasmid .Get your target fragment and plasmid backbone (Cas9 and pRH003 in our experiment) . Get CLB2 promoter fragment 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 each agar plate (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones were selected by colony PCR. Expanding culture 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 and release.

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

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

6. Obtain and analyze data. Draw the image of 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.