Difference between revisions of "Part:BBa K4165051"

(→‎Dry-lab Characterization)
(→‎Dry-lab Characterization)
Line 14: Line 14:
  
 
===Dry-lab Characterization===
 
===Dry-lab Characterization===
 +
<p style=" font-weight: bold; font-size:14px;">14) Top model</p>
 
<html>
 
<html>
−
<p><img src="https://static.igem.wiki/teams/4165/wiki/registry/dry-lab-modelling-pipeline.png" style="margin-left:200px;" alt="" width="500" /></p>
+
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1-bp/h1b.jpg" style="margin-left:200px;" alt="" width="500" /></p>
−
</html>
+
<html>
 +
<p style="text-align:center;">Figure 1.: 3D Model of H1A peptide Modelled by AlphaFold2.0 displayed in Pymol </p>
 +
 
 +
<html>
 +
<img src="https://static.igem.wiki/teams/4165/wiki/registry/dry-lab-modelling-pipeline.png" style="margin-left:200px;" alt="" width="500" /> <br>
 +
 
  
  
−
                    Figure 1. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.
+
<p style="text-align:center;">Figure 2. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.</p>
  
 
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
 
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
Line 33: Line 39:
 
<p style=" font-weight: bold; font-size:14px;"> 2) Structure Assessment </p>
 
<p style=" font-weight: bold; font-size:14px;"> 2) Structure Assessment </p>
 
<p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information:  
 
<p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information:  
−
<a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">modeling </a>.</p>
+
<a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling </a>.</p>
 
<p style=" font-weight: bold; font-size:14px;"> 3) Quality Assessment </p>
 
<p style=" font-weight: bold; font-size:14px;"> 3) Quality Assessment </p>
 
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.</p>
 
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.</p>
Line 44: Line 50:
  
 
<p style=" font-weight: bold; font-size:14px;">6) Ranking</p>
 
<p style=" font-weight: bold; font-size:14px;">6) Ranking</p>
−
<p>The docking results are ranked according to their PRODIGY results. For more information: (Link Docking page).</p>
+
<p>The docking results are ranked according to their PRODIGY results. For more information: <a href="https://2022.igem.wiki/cu-egypt/Docking.html"> Docking</a>.</p>
  
 
<p style=" font-weight: bold; font-size:14px;">7) Top Models</p>
 
<p style=" font-weight: bold; font-size:14px;">7) Top Models</p>
Line 51: Line 57:
 
<p style=" font-weight: bold; font-size:14px;">8) Alignment</p>
 
<p style=" font-weight: bold; font-size:14px;">8) Alignment</p>
 
<p>Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex which is the HtrA1-inhibitor complex to check whether they binded to the same site or not.</p>
 
<p>Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex which is the HtrA1-inhibitor complex to check whether they binded to the same site or not.</p>
−
<html>
+
 
−
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/picture10.png" style="margin-left:300px;" alt="" width="300" /></p>
+
<img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/picture10.png" style="margin-left:300px;" alt="" width="300" /></p>
−
</html>
+
 
  
  
−
                    Figure 2. Aligned structures of HtrA1 binding peptide 1 docked to HtrA1 and inhibitor docked to HtrA1.  
+
<p style="text-align:center;"> Figure 3. Aligned structures of HtrA1 binding peptide 1 docked to HtrA1 and inhibitor docked to HtrA1. </p>
 
<p style=" font-weight: bold; font-size:14px;">9) Linker length</p>
 
<p style=" font-weight: bold; font-size:14px;">9) Linker length</p>
 
<p>The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide which is between both C terminals, N terminals, C- and N- terminal, and N- and C-terminals.</p>
 
<p>The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide which is between both C terminals, N terminals, C- and N- terminal, and N- and C-terminals.</p>
Line 62: Line 68:
 
<p style=" font-weight: bold; font-size:14px;">10) Assembly</p>
 
<p style=" font-weight: bold; font-size:14px;">10) Assembly</p>
 
<p>After settling on the linkers lengths, now we will proceed to the assembly step of the whole system which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.</p>
 
<p>After settling on the linkers lengths, now we will proceed to the assembly step of the whole system which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.</p>
−
<html>
 
−
<p>a<img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/ninhiibitor31-clamp.png" style="margin-left:50px;" alt="" width="150" />b<img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1-bp/h1b.jpg" style="margin-left:50px;" alt="" width="150" />c<img src="https://static.igem.wiki/teams/4165/wiki/q8iub5-trrosetta-model3.png" style="margin-left:50px;" alt="" width="150" /></p>
 
−
</html>
 
  
−
    Figure 3. a) Seed_GGSGGGGG_seed clamp b) HTRA Binding Peptide 1 c)WAP-four disulfide core domain 13 serine protease inhibitor.
+
<p>a<img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/ninhiibitor31-clamp.png" style="margin-left:50px;" alt="" width="150"/> b<img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/htra1-bp/h1b.jpg" style="margin-left:50px;" alt="" width="150" />c<img src="https://static.igem.wiki/teams/4165/wiki/q8iub5-trrosetta-model3.png" style="margin-left:50px;" alt="" width="150" /></p>
 +
 
 +
 
 +
Figure 4. a) Seed_GGSGGGGG_seed clamp b) HTRA Binding Peptide 1 c)WAP-four disulfide core domain 13 serine protease inhibitor.
 
<p style=" font-weight: bold; font-size:14px;">11) Structure Assessment</p>
 
<p style=" font-weight: bold; font-size:14px;">11) Structure Assessment</p>
−
<p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link modelling page).</p>
+
<p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (<a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling </a>).</p>
  
 
<p style=" font-weight: bold; font-size:14px;">12) Quality Assessment </p>
 
<p style=" font-weight: bold; font-size:14px;">12) Quality Assessment </p>
−
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.</p>
+
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (<a href="https://2022.igem.wiki/cu-egypt/software.html">Software </a>) under the name of Modric.</p>
  
  
Line 77: Line 83:
 
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Abu Trika.</p>
 
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Abu Trika.</p>
 
<p style=" font-weight: bold; font-size:14px;">Table 1. quality assessment parameters of switch 31.</p>
 
<p style=" font-weight: bold; font-size:14px;">Table 1. quality assessment parameters of switch 31.</p>
−
<html>
+
 
−
<style>
+
 
−
table, th, td {
+
−
  border:1px solid black; margin-left:auto;margin-right:auto;
+
−
}
+
−
</style>
+
−
<body>
+
 
<table style="width:65%">
 
<table style="width:65%">
 
<table>
 
<table>
Line 105: Line 106:
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
</body>
+
 
−
</html>
+
 
<p style=" font-weight: bold; font-size:14px;">14) Alignment</p>
 
<p style=" font-weight: bold; font-size:14px;">14) Alignment</p>
 
<p>The docked structures are then aligned and compared to the basic parts which are docked with protein of interest (HtrA1). The structures with least RMSD are chosen.</p>
 
<p>The docked structures are then aligned and compared to the basic parts which are docked with protein of interest (HtrA1). The structures with least RMSD are chosen.</p>
−
<html>
+
 
−
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/switch31.png" style="margin-left:300px;" alt="" width="250" /></p>
+
<img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/switch31/switch31.png" style="margin-left:300px;" alt="" width="250" />
−
</html>
+
 
−
                                        Figure 3. Structure of top model of switch 31.
+
          <p style="text-align:center;">Figure 5. Structure of top model of switch 31.</p>
 
<p style=" font-weight: bold; font-size:14px;">Table 2. RMSD calculated from alignment of switch 31 and its basic parts.</p>
 
<p style=" font-weight: bold; font-size:14px;">Table 2. RMSD calculated from alignment of switch 31 and its basic parts.</p>
−
<html>
+
 
 
<style>
 
<style>
 
table, th, td {
 
table, th, td {

Revision as of 20:54, 13 October 2022


HtrA1 Switch 31

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), H1A (BBa_K4165000), GS Linker (BBa_K4165066), seed peptide (BBa_K4165012), GS Linker (BBa_K4165019), seed peptide (BBa_K4165012), GS Linker (BBa_K4165066), WAP inhibitor (BBa_K4165008), and T7 terminator (BBa_K731721).

Usage and Biology

Switch 31 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 PDZ peptide, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 589
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 572
    Illegal AgeI site found at 308
  • 1000
    COMPATIBLE WITH RFC[1000]

Dry-lab Characterization

14) Top model

Figure 1.: 3D Model of H1A peptide Modelled by AlphaFold2.0 displayed in Pymol


Figure 2. A figure which dsecribes our Dry-Lab Modelling Pipeline. By team CU_Egypt 2022.

Modeling

The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta.

Switch construction Pipeline

1) Modelling

Since our parts do not have experimentally acquired structures, we have to model them. This approach is done using both denovo modelling (ab initio) and template-based modelling. For modelling small peptides of our system we used AppTest and Alphafold.

2) Structure Assessment

In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: Modeling .

3) Quality Assessment

Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.

4) Filtering

We take the top ranked models from the previous steps.

5) Docking

The top models of inhibitor and HTRA Binding Peptide are docked with HtrA1 (BBa_K4165004).

6) Ranking

The docking results are ranked according to their PRODIGY results. For more information: Docking.

7) Top Models

The results that came out from PRODIGY are ranked and top models are chosen to proceed with to the next step. For more information: (Link Docking page).

8) Alignment

Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex which is the HtrA1-inhibitor complex to check whether they binded to the same site or not.

Figure 3. Aligned structures of HtrA1 binding peptide 1 docked to HtrA1 and inhibitor docked to HtrA1.

9) Linker length

The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide which is between both C terminals, N terminals, C- and N- terminal, and N- and C-terminals.

10) Assembly

After settling on the linkers lengths, now we will proceed to the assembly step of the whole system which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.

a bc

Figure 4. a) Seed_GGSGGGGG_seed clamp b) HTRA Binding Peptide 1 c)WAP-four disulfide core domain 13 serine protease inhibitor.

11) Structure Assessment

In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Modeling ).

12) Quality Assessment

Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Software ) under the name of Modric.

13) Ranking

Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Abu Trika.

Table 1. quality assessment parameters of switch 31.

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
2 2.18 1.55 89.84 1.56 0.195965 0.196775

14) Alignment

The docked structures are then aligned and compared to the basic parts which are docked with protein of interest (HtrA1). The structures with least RMSD are chosen.

Figure 5. Structure of top model of switch 31.

Table 2. RMSD calculated from alignment of switch 31 and its basic parts.

average RMSD from free HtrA binding peptide1 average RMSD from docked HtrA binding peptide1 RMSD from free seed peptide
1.367 1.674 4.466