Difference between revisions of "Part:BBa K3506009"
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<b><font size="3">Experimental approach</font></b>
 
<b><font size="3">Experimental approach</font></b>
  
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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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1. Construct recombinant plasmid.Get Cas9 from pRH003.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.  
 
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.
 
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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4. Synchronize S. cerevisiae cells. Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize yeast cells.
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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.  
+
5. Release 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.
 
6. Obtain and analyze data. Draw the image of Cas9 protein levels over time.

Revision as of 17:12, 21 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 level of Cas9 change with the cell cycle. Finally, Cas9 expression began in the S phase and reached its peak in the G2/M phase.


Usage

This part can be used to control Cas9 expression level with yeast cell cycle. If you want to control the expression of Cas9 during cell division, you can place this part on the 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 812
    Illegal BglII site found at 1607
    Illegal BamHI site found at 1901
    Illegal XhoI site found at 203
    Illegal XhoI site found at 2407
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1639
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 730
    Illegal BsaI.rc site found at 1972
    Illegal BsaI.rc site found at 3328
    Illegal BsaI.rc site found at 3751
    Illegal BsaI.rc site found at 3763
    Illegal BsaI.rc site found at 4630
    Illegal SapI.rc site found at 3435
    Illegal SapI.rc site found at 4017
    Illegal SapI.rc site found at 4032


Design and Properties

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 (BBa_K206000).


Experimental approach

1. Construct recombinant plasmid.Get Cas9 from pRH003.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. Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize yeast cells.

5. Release 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]Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121):819-23. doi: 10.1126/science.1231143. Epub 2013 Jan 3. PMID: 23287718; PMCID: PMC3795411.