Difference between revisions of "Part:BBa K3506007"
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<b><font size="3">Biology and Usage</font></b>
 
<b><font size="3">Biology and Usage</font></b>
  
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<i>CLB2</i> promoter is the promoter of the G2/mitotic-specific Cyclin-B2 (Clb2) which accumulates steadily during G2 and is rapidly destroyed at mitosis. Clb2 interacts with the Cdc28 protein kinase to form maturation-promoting factor (MPF) and is critical for the control of cell cycle at  G2/M (mitosis) transition. Previous studies have revealed that <i>CLB2</i> promoter can regulate the transcription of its downstream gene periodically. Under the control of <i>CLB2</i> promoter, the downstream gene starts to transcribe in S phase, reaches a peak at the G2/M transition, and is rapidly degraded at the end of M phase (Figure 1).
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<i>CLB2</i> promoter is the promoter of the G2/mitotic-specific Cyclin-B2 (Clb2) which accumulates steadily during G2 and is rapidly destroyed at mitosis. Clb2 interacts with the Cdc28 protein kinase to form the maturation-promoting factor (MPF) and is critical for the control of the cell cycle at  G2/M (mitosis) transition. Previous studies have revealed that <i>CLB2</i> promoter can regulate the transcription of its downstream gene periodically. Under the control of <i>CLB2</i> promoter, the downstream gene starts to transcribe in the S phase, reaches a peak at the G2/M transition, and is rapidly degraded at the end of the M phase (Figure 1).
  
 
This part can be used to couple the transcription of the target gene with the yeast cell cycle. To express target genes during cell division, one can place <i>CLB2</i> promoter sequence before the gene on a yeast plasmid.
 
This part can be used to couple the transcription of the target gene with the yeast cell cycle. To express target genes during cell division, one can place <i>CLB2</i> promoter sequence before the gene on a yeast plasmid.
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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 <i>Saccharomyces cerevisiae</i> S288C by PCR. Ligate the fragments by in-fusion cloning.
 
1. Construct recombinant plasmid .Get your target fragment and plasmid backbone (Cas9 and pRH003 in our experiment). Get CLB2 promoter fragment from <i>Saccharomyces cerevisiae</i> S288C by PCR. Ligate the fragments by in-fusion cloning.
  
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2. Transform the product (2.5μL) into <i>E. coli</i> 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.
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2. Transform the product (2.5μL) into <i>E. coli</i> 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 sequences.
  
 
3. Linearize the plasmids with XhoI and transform them (5-10ng) into <i>Saccharomyces cerevisiae</i> BY4741. Grow cells on SD-ura plate and incubate at 30℃ for 3 days. Monoclones were selected by colony PCR and sequencing.
 
3. Linearize the plasmids with XhoI and transform them (5-10ng) into <i>Saccharomyces cerevisiae</i> BY4741. Grow cells on SD-ura plate and incubate at 30℃ for 3 days. Monoclones were selected by colony PCR and sequencing.
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Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize and release yeast cells.
 
Several methods (Alpha Factor、Nutrient Depletion、Hydroxyurea) can be used to synchronize and release yeast cells.
  
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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.
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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 a negative control. Don’t forget to select the internal reference.
  
 
6. Obtain and analyze data. Draw the image of protein levels over time.
 
6. Obtain and analyze data. Draw the image of protein levels over time.

Revision as of 10:01, 27 October 2020


CLB2 promoter

CLB2 promoter is the putative promoter sequence of the G2/mitotic-specific Cyclin-B2 (Clb2) encoding gene which promotes its transcription in late S and G2/M in the yeast cell cycle. Its sequence is taken from -829nt to ATG codon in the genome of Saccharomyces cerevisiae BY4741.This part is used to control the periodic expression of Cas9 so that we can couple the expression of Cas9 with cell division.


Biology and Usage

CLB2 promoter is the promoter of the G2/mitotic-specific Cyclin-B2 (Clb2) which accumulates steadily during G2 and is rapidly destroyed at mitosis. Clb2 interacts with the Cdc28 protein kinase to form the maturation-promoting factor (MPF) and is critical for the control of the cell cycle at 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 transcribe in the S phase, reaches a peak at the G2/M transition, and is rapidly degraded at the end of the M phase (Figure 1).

This part can be used to couple the transcription of the target gene with the yeast cell cycle. To express target genes during cell division, one can place CLB2 promoter sequence before the gene 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 XhoI site found at 203
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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 sequences.

3. Linearize the plasmids with XhoI 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 a 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]Mendenhall, M. D., & Hodge, A. E. (1998). Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiology and molecular biology reviews: MMBR, 62(4), 1191–1243.

[2]Trcek, T., Larson, D. R., Moldón, A., Query, C. C., & Singer, R. H. (2011). Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast. Cell, 147(7), 1484–1497.

[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 (Clifton, N.J.), 761, 173–200.