Difference between revisions of "Part:BBa K3506007"

Revision as of 13:36, 19 October 2020

CLB2 promoter

This part is the CLB2 promoter which is -829 to -365 nt relative to CLB2 ATG on the genome of Saccharomyces cerevisiae BY4741. CLB2 promoter is the promoter of the G2/mitotic-specific cyclin-2(CLB2) which accumulates steadily during G2 and is abruptly destroyed at mitosis. CLB2 interacts with the CDC2 protein kinase to form maturation-promoting factor(MPF) and is essential for the control of the cell cycle at the G2/M (mitosis) transition.Previous studies have revealed that CLB2 promoter can regulate the transcription of its downstream gene periodically. Under the control of CLB2 promoter, the downstream gene starts to transcript in the S phase, reaches a peak at the G2/M transition, and rapidly degrades at the end of the M phase.

Biology and Usage

This part can be used to couple the transcription of the target gene with yeast cell cycle.CLB2 promoter is a promoter which promotes transciption in late S and G2/M of the yeast cell cycle .If you want to express target gene during cell division,you can place the Clb2 promoter sequence before your gene sequence on the yeast plasmid.

Sequence and Features

Experimental approach

1. Construct recombinant plasmid.Get your target fragment and plasmid backbone (Cas9 and pRH003 in our experiment).Get CLB2 promoter fragment 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 and release. Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize and release yeast cells.

5. 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.https://doi.org/10.1007/978-1-61779-273-1_5

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