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| − | 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.
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| − | 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.
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| − | 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.
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| − | 4. Synchronize S. cerevisiae cells and release.
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| − | Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize and release yeast cells.
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| − | 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.
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| − | 6. Obtain and analyze data.Draw the image of Cas9 protein levels over time.
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| | ===Applications of BBa_K3506007=== | | ===Applications of BBa_K3506007=== |
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| − | [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.
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| − | [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.
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| − | [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
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| − | [4] Manukyan, A. , Abraham, L. , Dungrawala, H. , & Schneider, B. L. . (2011). Synchronization of yeast. Methods in Molecular Biology, 761(761), 173.
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