Difference between revisions of "Part:BBa K4165202"

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<partinfo>BBa_K4165202 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165202 SequenceAndFeatures</partinfo>
  
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===References===
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===Dry Lab===
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1-Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.
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<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
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MM2 is modeled by AlphaFold2, ITASSER and TrRosetta, best model obtained from TrRosetta.  
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/trim-g4s3-docs.png" style="margin-left:150px;" alt="" width="500" /></p>
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                            Figure 1.: Predicted 3D structure of our fusion protein tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS.
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<p style=" font-weight: bold; font-size:13px;"> Table 1: Quality assessment parameters of tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS. model. </p>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/trim-g4s3-docsqa.png" style="margin-left:75px;" alt="" width="700"
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/trim-doc-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/trim-doc-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
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                                     Figure 1. Transformed plate of His Trim21 (L) Doc + pGS-21a  
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                                     Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a  
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector </p>
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector </p>
 
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-trim-doc-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-trim-doc-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
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                                     Figure 2. Transformed plate of His Trim21 (L) Doc + pJET  
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                                     Figure 3. Transformed plate of His Trim21 (L) Doc + pJET  
 
<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for His Trim21 (L) DOC </p>
 
<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for His Trim21 (L) DOC </p>
 
<html>
 
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<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/trim-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/trim-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
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Figure 3. This graph shows a significant difference between chemical lysis and sonication for His Trim21 (L) DOC, after we had the results we optimized our protocol to use sonication for His Trim21 (L) DOC
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Figure 4. This graph shows a significant difference between chemical lysis and sonication for His Trim21 (L) DOC, after we had the results we optimized our protocol to use sonication for His Trim21 (L) DOC
 +
 
 +
===References===
 +
1-Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 19:38, 11 October 2022


Trim-(GGGGS)3-Docs

This parts code for the trim21 E3 ligase having his PRYSPRY domain truncated (BBa_K3396007), fused to type 1 Dockerin module derived from Clostridium thermocellum cellulosome scaffoldin using Glycine serine flexible linker repeated three times to maintain part flexibility needed during target ubiquitination.

source

Synthetic Fusion protein

Sequence and Features

sequence was optimized for E.coli expression


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 535

Dry Lab

Modeling

MM2 is modeled by AlphaFold2, ITASSER and TrRosetta, best model obtained from TrRosetta. 


                            Figure 1.: Predicted 3D structure of our fusion protein tTrim21-(G4S)3-DocS.


Table 1: Quality assessment parameters of tTrim21-(G4S)3-DocS. model.



WetLab Results

Transformation of His Trim21 (L) Doc in BL-21 using pGS-21a 

                                   Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a

Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector 

                                   Figure 3. Transformed plate of His Trim21 (L) Doc + pJET

Comparison between chemical lysis and sonication for His Trim21 (L) DOC

Figure 4. This graph shows a significant difference between chemical lysis and sonication for His Trim21 (L) DOC, after we had the results we optimized our protocol to use sonication for His Trim21 (L) DOC

References

1-Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.