Difference between revisions of "Part:BBa K4165031"

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<h2>Sequence and Features</h2>
 
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<partinfo>BBa_K4165031 SequenceAndFeatures</partinfo>
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===Dry-lab Characterization===
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<p><img src="https://static.igem.wiki/teams/4165/wiki/registry/dry-lab-modelling-pipeline.png" style="margin-left:200px;" alt="" width="500" /></p>
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                    Figure 1. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.
  
 
===Dry lab===
 
===Dry lab===

Revision as of 17:39, 13 October 2022


HtrA1 switch 11

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), SPINK8 Inhibitor (BBa_K4165010), TD28rev (BBa_K4165006), WWW (BBa_K4165007), and T7 terminator (BBa_K731721).


Usage and Biology

Switch 11 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 peptide binding PDZ, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 upon clamp binding to the target protein requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.

The TD28REV and WWW peptides considered as tau binding peptides are proved experimentally to bind with tau inhibit the aggregations of tau aggregations respectively. The H1A peptide was also proven to bind with the PDZ of HtrA1 experimentally. The last part is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, it will act and inhibit the Protein. The whole construction was similarly proved from literature. The process of assembly of the whole switch was done according to both CAPRI and NCBI protocols.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 528
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Dry-lab Characterization


                   Figure 1. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.

Dry lab

Modeling

The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta with a score of 4 out of 6 according to our quality assessment code.

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 3.76 1.5 95.17 0.69 -0.31007 -1.21262 


                              Figure 1. The 3D structure of switch 11 visualized by Pymol.