Difference between revisions of "Part:BBa K3734019"
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                 <p>After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.</p>>
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                 <p>After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.</p>
 
                 <p>We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.
 
                 <p>We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.
 
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system</p>
 
                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system</p>
 
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                     <img width="400px" src="https://2021.igem.org/wiki/images/1/1f/T--CSU_CHINA--tupian16.png"></p>
 
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.3 ERK phosphorylation changes with different insulin treatment</p>
 
                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.3 ERK phosphorylation changes with different insulin treatment</p>
 
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                     <img width="400px" src="https://2021.igem.org/wiki/images/5/54/T--CSU_CHINA--tupian49.png"></p>
 
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.4 ERK phosphorylation changes with different insulin treatment</p>
 
                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.4 ERK phosphorylation changes with different insulin treatment</p>
 
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Revision as of 12:05, 20 October 2021

TetR-ELK

TetR is a kind of protein that can find and combined with TRE DNA structure domain.ELK is a kind of protein that can activate downstream gene expression after being regulated by phosphorylation

TetR-ELK

TetR can recognize and combine with TRE, it is a important part of Tet-off system. Without tetracycline, TetR can combine with TRE. With the presence of tetracycline, the combination of TetR and TRE will be blocked and the Tet-off system will be shut down. ELK is generally dormant, when INSR receive insulin and activate MAPK, ELK will be phophated and activated, then ELK is able activate expression of target gene downstream TRE.

1.Pattern diagram

Fig.1 The model diagram TetR-ELK

2.Experiment

2.1 Method

After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.

We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.

2.2 Result


Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system


Fig.3 ERK phosphorylation changes with different insulin treatment


Fig.4 ERK phosphorylation changes with different insulin treatment

3.Caution

Despite the length and the complication of phosphorylation pathway, the phosphorylation pathway of protein is a very short process, it is usually completed within minutes even seconds. The time of cracking the cells and collecting protein must be controlled precisely when detecting

Reference：

[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 621
    Illegal NgoMIV site found at 759
    Illegal AgeI site found at 999
  • 1000
    COMPATIBLE WITH RFC[1000]