Difference between revisions of "Part:BBa K3732000"
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<partinfo>BBa_K3732000 parameters</partinfo>
 
<partinfo>BBa_K3732000 parameters</partinfo>
 
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==Characterization==
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<html>
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<figure>
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        <img src="https://2021.igem.org/wiki/images/a/ad/T--FAFU-CHINA--20-2.jpeg" width="45%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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</figure>
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</html>
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<p>
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The PCR results showed that we successfully assembled BBa_K3732002, BBa_K3732000 and BBa_K3732001 together.
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</p>
  
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==Contribution from iGEM21-FAFU-China==
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==Contribution From FAFU-China 2021==
  
 
===LOV2===
 
===LOV2===
  
 
<p>
 
<p>
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The light-receptor domain LOV2 binds flavin mononucleotide (FMN) as a cofactor, which is the main photoactivated compound. Excitation of the FMN cofactors by blue light results in the formation of a bond between FMN and cysteine in the LOV2 domain. This induces structural rearrangement of the LOV2 domain, leading to the unfolding of the α -helix at the carboxyl terminal.
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The light-receptor domain LOV2 binds flavin mononucleotide (FMN) as a cofactor, which is the main photoactivated compound. Excitation of the FMN cofactors by blue light results in the formation of a bond between FMN and cysteine in the LOV2 domain. This induces structural rearrangement of the LOV2 domain, leading to the unfolding of the α -helix at the carboxyl terminal.<sup>「1」</sup>
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<sup>「1」</sup>
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</p>
 
</p>
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<html>
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<figure>
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        <img src="https://2021.igem.org/wiki/images/6/6e/T--FAFU-CHINA--7-8.png" width="45%" style="float:center">
 +
        <figcaption>
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        <p style="font-size:1rem">
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        Protein structure of the LOV2 domain and light-induced structural changes during the photocycle.
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        </p>
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        </figcaption>
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</figure>
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</html>
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<p>
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The green strip shows the β -folding of the LOV2 core, while the rod-like structure highlights three key residues in FMN and signal transduction, Cys966, Phe1008 and Gln1029.
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</p>
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<html>
 +
<figure>
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        <img src="https://2021.igem.org/wiki/images/4/4e/T--FAFU-CHINA--7-13.png" width="45%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        LOV2 activation stage and experimental test time
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        </p>
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        </figcaption>
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</figure>
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</html>
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<p>
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The dynamics of the chromophore mainly occur on the ultra-fast time scale, and the protein matrix changes occur after this, and the time constant of the entire Jα helix unwinding is 313µs. Compared with the light state Jα helix, corresponding to the residues of the whole protein including the Jα helix and the Jα helix alone, the RMSD vs. time is plotted as shown below.
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</p>
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<html>
 +
<figure>
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        <img src="https://2021.igem.org/wiki/images/d/df/T--FAFU-CHINA--7-14.png" width="45%" style="float:center">
 +
        <figcaption>
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        <p style="font-size:1rem">
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        Molecular dynamics simulation shows the stability of the Jα helix
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        </p>
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        </figcaption>
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</figure>
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</html>
  
 
===cODC1===
 
===cODC1===
  
 
<p>
 
<p>
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In ODC, the carboxy-terminal 37 amino acids (cODC) function as a degron that is directly  recognized by the proteasome.
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In ODC, the carboxy-terminal 37 amino acids (cODC) function as a degron that is directly  recognized by the proteasome.<sup>「2」</sup>
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<sup>「2」</sup>
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</p>
 
</p>
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<html>
 
<figure>
 
<figure>
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         <img src="" width="45%" style="float:center">
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         <img src="https://2021.igem.org/wiki/images/e/e1/T--FAFU-CHINA--8-1.png" width="45%" style="float:center">
 
         <figcaption>
 
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         </figcaption>
 
         </figcaption>
 
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</figure>
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<p>
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By fusing cODC1 to the end of LOV2, we have an element that uses light signals to regulate protein abundance.
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</p>
 
<p>
 
<p>
 
When LOV2 is activated by light，which makes the cODC1 degron accessible for recognition by the  proteasome  and  ubiquitin - independent degradation of the whole construct.<sup>「1」</sup>
 
When LOV2 is activated by light，which makes the cODC1 degron accessible for recognition by the  proteasome  and  ubiquitin - independent degradation of the whole construct.<sup>「1」</sup>
 
</p>
 
</p>

Latest revision as of 19:02, 19 October 2021


photosensitive degron (psd) module.

The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fi ne-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).

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]


Characterization

The PCR results showed that we successfully assembled BBa_K3732002, BBa_K3732000 and BBa_K3732001 together.

Contribution From FAFU-China 2021

LOV2

The light-receptor domain LOV2 binds flavin mononucleotide (FMN) as a cofactor, which is the main photoactivated compound. Excitation of the FMN cofactors by blue light results in the formation of a bond between FMN and cysteine in the LOV2 domain. This induces structural rearrangement of the LOV2 domain, leading to the unfolding of the α -helix at the carboxyl terminal.「1」

Protein structure of the LOV2 domain and light-induced structural changes during the photocycle.

The green strip shows the β -folding of the LOV2 core, while the rod-like structure highlights three key residues in FMN and signal transduction, Cys966, Phe1008 and Gln1029.

LOV2 activation stage and experimental test time

The dynamics of the chromophore mainly occur on the ultra-fast time scale, and the protein matrix changes occur after this, and the time constant of the entire Jα helix unwinding is 313µs. Compared with the light state Jα helix, corresponding to the residues of the whole protein including the Jα helix and the Jα helix alone, the RMSD vs. time is plotted as shown below.

Molecular dynamics simulation shows the stability of the Jα helix

cODC1

In ODC, the carboxy-terminal 37 amino acids (cODC) function as a degron that is directly recognized by the proteasome.「2」

By fusing cODC1 to the end of LOV2, we have an element that uses light signals to regulate protein abundance.

When LOV2 is activated by light，which makes the cODC1 degron accessible for recognition by the proteasome and ubiquitin - independent degradation of the whole construct.「1」