Difference between revisions of "Part:BBa K3506010"
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<partinfo>BBa_K3506010 short</partinfo>
 
<partinfo>BBa_K3506010 short</partinfo>
  
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Over the four mitotic cyclins, cyclin b2 has the most important role in the yeast Saccharomyces cerevisiae, which is sufficient to trigger all essential functions of cyclin-dependent kinases in mitosis by itself. Research shows that a Clb2 KEN box can only degrade targeted fusion protein with the help of the first 124 amino acids of Clb2. So during MA and G1, a sequence of Clb2 124aa containing the KEN box and the destruction box act together to regulate proteolysis of Clb2 fusion protein.[1]
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Of the four mitotic cyclins, Clb2 has the most important role in <i>Saccharomyces cerevisiae</i>. It alone is sufficient to trigger all essential functions of cyclin-dependent kinases in mitosis. Research shows that the first 124 amino acids of Clb2 can promotes the degradation of it because it contain the D-Box and KEN-Box,which are the recognition site by E3 ubiquitin ligase APC (Anaphase-promoting complex).
  
 
<b><font size="3">Biology and Usage</font></b>
 
<b><font size="3">Biology and Usage</font></b>
  
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Clb2 N124aa is the first 124 amino acids of Clb2. In Saccharomyces cerevisiae BY4741, its function is to help the degradation of CLB2 during M and G1 phase.
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Clb2 N124aa is the first 124 amino acids of Clb2. In <i>Saccharomyces cerevisiae</i> BY4741, its function is to help the degradation of CLB2 during M and G1 phase.
 
In our project, it is used to degrade Cas9 at a proper speed, thus achieve the goal to switch off our system.
 
In our project, it is used to degrade Cas9 at a proper speed, thus achieve the goal to switch off our system.
 
This part can be fused to another protein to increase the degradation rate of it.
 
This part can be fused to another protein to increase the degradation rate of it.
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<b><font size="3">Properties</font></b>
 
<b><font size="3">Properties</font></b>
  
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This part was fused to GFP at the N-terminal and transformed into Saccharomyces cerevisiae BY4741. After induced by galactose for 10 hours, Saccharomyces cerevisiae BY4741 were transferred to the SC-ura(glucose) medium. Then the fluorescence intensity was measured every 30 mins by flow cytometry for 5 hours. After data collection, the protein degradation curve characterized by fluorescence intensity was processed and plotted. As shown in figure below, compared with control group, the Clb2N124aa and GFP fusion protein degrades much faster.
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This part was fused to GFP at the N-terminal and transformed into <i>Saccharomyces cerevisiae</i> BY4741. After induced by galactose for 10 hours, <i>Saccharomyces cerevisiae</i> BY4741 were transferred to the SC-ura(glucose) medium. Then the fluorescence intensity was measured every 30 mins by flow cytometry for 5 hours. After data collection, the protein degradation curve characterized by fluorescence intensity was processed and plotted. As shown in figure below, compared with control group, the Clb2 N124aa and GFP fusion protein degrades much faster.
  
 
[[Image:T--BNU-China--K3506010.png|700px|thumb|center|Fig. 1 Fluorescence intensity by time]]
 
[[Image:T--BNU-China--K3506010.png|700px|thumb|center|Fig. 1 Fluorescence intensity by time]]
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<b><font size="3">Expenrimental approach</font></b>
 
<b><font size="3">Expenrimental approach</font></b>
  
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1、Clb2 N124aa was PCRed down from the brewer's yeast genome using specific primers.
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1.Clb2 N124aa was amplified by PCR from the <i>Saccharomyces cerevisiae</i> genome using specific primers.
  
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2、The double enzyme-cut pYES2 plasmid backbone (containing Gal1 promoter), Clb2 N124aa, and GFP fragments were cloned into the target plasmid in the order of pYES2 plasmid backbone (containing Gal1 promoter)-Clb2 N124aa-GFP fragment by infusion cloning relying on the homology arm. (Experimental group of target plasmids)
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2.The pYES2 plasmid (containing <i>GAL1</i> promoter), Clb2 N124aa, and GFP fragment were connected by in-fusion cloning as experimental group.
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The target plasmid was cloned in the order of pYES2 plasmid backbone (containing Gal1 promoter) and GFP fragment after double enzymatic cutting by infusion cloning relying on the homology arm. (Control target plasmid)
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3、Both plasmids were transformed into E. coli DH5α-sensing state cells, respectively.
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3.Both experimental group and control group were transformed into <i>E. coli</i> DH5α.
  
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4、The transformed E. coli were incubated in 20 mL of YPD-amphenicol (100 mg/ml) in an incubator at 37°C, 180 rpm for 12 hours.
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4.The transformed <i>E. coli</i> were grown in 20 mL of LB-amphenicol (100 mg/ml) in an incubator at 37°C, 180 rpm for 12 hours.
  
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5、The experimental group aspirated 1 ml of bacterial solution coated into YPD-amphenicillin (100 mg/ml) medium and incubated at 37°C for 12 h in the incubator.
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5.1 ml of bacterial solution was inoculated into LB-amphenicillin (100 mg/ml) medium and incubated at 37°C for 12 hours in the incubator.
  
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6、Search for single colonies in YPD-amphenicol (100 mg/l) medium for colony PCR to verify the successful transfer of the target plasmids.
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6.Screen for single colonies in LB-amphenicol (100 mg/l) medium and perform colony PCR to verify the successful transformation.
  
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7、Select the single colony successfully transformed into sensory winery cells and incubate in SD-ura medium at a constant temperature of 30°C for 14-16h.
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7.Select the single colony successfully transformed and incubate overnight in a shaker.
  
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8、Single colonies were sought in SD-ura medium for colony PCR to verify successful transfer to the target plasmid.
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8.Extract the plasmids from <i>E.coli</i> and linearize by single enzyme.
  
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9、Selected experimental and control groups of single-colony bacterial solution were coated in SD-ura medium and incubated at 37°C for 14-16h in the incubator.
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9.Transform the plasmids into competent yeast cell and grow it in SC-ura(glucose) medium at a constant temperature of 30°C for 2 days.
  
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10、When glucose was depleted, galactose was added to the culture medium every 2h to induce the expression of GFP. In the control group, GFP was expressed in the presence of galactose in the environment.
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10.Screen single colonies by colony PCR to verify successful transformation.
  
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11、Add glucose to the medium to a final concentration of 2% to remove the induction of galactose.
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11.Selected colonies were inoculated into the SC-ura(glucose) medium and grown at 30°C for 14-16h in the incubator.
  
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12、Culture samples were removed before (t=0) and at specified times after the addition of glucose, and cells were loaded for fluorescence microscopy.
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12.Transfer the yeast cell to the SC-ura(galactose) medium.
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13.After 10 hours, transfer the yeast cell to the SC-ura(glucose) medium. Then measure the fluorescence intensity every one and a half hours by flow cytometry for 5 hours.
  
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13、Autofluorescence was determined by quantification of non-expressing GFP by subtracting the mean value of non-expressing GFP cells.
 
  
  

Revision as of 20:36, 26 October 2020


Clb2 N124aa

Of the four mitotic cyclins, Clb2 has the most important role in Saccharomyces cerevisiae. It alone is sufficient to trigger all essential functions of cyclin-dependent kinases in mitosis. Research shows that the first 124 amino acids of Clb2 can promotes the degradation of it because it contain the D-Box and KEN-Box,which are the recognition site by E3 ubiquitin ligase APC (Anaphase-promoting complex).

Biology and Usage

Clb2 N124aa is the first 124 amino acids of Clb2. In Saccharomyces cerevisiae BY4741, its function is to help the degradation of CLB2 during M and G1 phase. In our project, it is used to degrade Cas9 at a proper speed, thus achieve the goal to switch off our system. This part can be fused to another protein to increase the degradation rate of it.


Properties

This part was fused to GFP at the N-terminal and transformed into Saccharomyces cerevisiae BY4741. After induced by galactose for 10 hours, Saccharomyces cerevisiae BY4741 were transferred to the SC-ura(glucose) medium. Then the fluorescence intensity was measured every 30 mins by flow cytometry for 5 hours. After data collection, the protein degradation curve characterized by fluorescence intensity was processed and plotted. As shown in figure below, compared with control group, the Clb2 N124aa and GFP fusion protein degrades much faster.

Fig. 1 Fluorescence intensity by time

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Expenrimental approach

1.Clb2 N124aa was amplified by PCR from the Saccharomyces cerevisiae genome using specific primers.

2.The pYES2 plasmid (containing GAL1 promoter), Clb2 N124aa, and GFP fragment were connected by in-fusion cloning as experimental group.

3.Both experimental group and control group were transformed into E. coli DH5α.

4.The transformed E. coli were grown in 20 mL of LB-amphenicol (100 mg/ml) in an incubator at 37°C, 180 rpm for 12 hours.

5.1 ml of bacterial solution was inoculated into LB-amphenicillin (100 mg/ml) medium and incubated at 37°C for 12 hours in the incubator.

6.Screen for single colonies in LB-amphenicol (100 mg/l) medium and perform colony PCR to verify the successful transformation.

7.Select the single colony successfully transformed and incubate overnight in a shaker.

8.Extract the plasmids from E.coli and linearize by single enzyme.

9.Transform the plasmids into competent yeast cell and grow it in SC-ura(glucose) medium at a constant temperature of 30°C for 2 days.

10.Screen single colonies by colony PCR to verify successful transformation.

11.Selected colonies were inoculated into the SC-ura(glucose) medium and grown at 30°C for 14-16h in the incubator.

12.Transfer the yeast cell to the SC-ura(galactose) medium.

13.After 10 hours, transfer the yeast cell to the SC-ura(glucose) medium. Then measure the fluorescence intensity every one and a half hours by flow cytometry for 5 hours.


References

[1]Hendrickson, C., Meyn, M. A., 3rd, Morabito, L., & Holloway, S. L. (2001). The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition. Current biology : CB, 11(22), 1781–1787.