Difference between revisions of "Part:BBa K4165038"
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This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGSGS linker (BBa_J18921), seed peptide (BBa_K4165012), GGSGGGGG linker (BBa_K4165019), seed peptide (BBa_K4165012), GSGSGS linker (BBa_J18921), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).
 
This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGSGS linker (BBa_J18921), seed peptide (BBa_K4165012), GGSGGGGG linker (BBa_K4165019), seed peptide (BBa_K4165012), GSGSGS linker (BBa_J18921), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).
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===Source===
 
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Synthesized
 
  
 
===Usage and Biology===
 
===Usage and Biology===
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Switch 18 is used to mediate the activity of HTRA1. 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 affinity clamp for tau and beta-amyloid binding. These clamps are used for stabilizing the inhibitor away from the active site. These two domains (inhibitor and affinity clamp connected with linker1). Additionally, (H1A) binding peptide bound to the PDZ domain and connected to the affinity clamp on the other side with linker3.
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Switch 18 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.
  
 
<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
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===Dry Lab===
 
===Dry Lab===
 
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
 
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
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TRrosetta models this composite part with a score of 4 out of 6 according to our quality assessment code (you can find the python script file on the programming club page with further explanation of how you can optimize it to your needs).
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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.
  
 
<html>
 
<html>
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</html>
 
</html>
  
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                          Figure 1. The 3D structure of switch 18 modeled by TRrosetta. Red: Tau binding peptides,  
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                    Figure 1. The 3D structure of switch 18 modeled by TRrosetta. Red: Tau binding peptides,  
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                                          blue: H1A peptide, cyan: inhibitor, and green: linkers
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                                  blue: H1A peptide, cyan: inhibitor, and green: linkers
  
  
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</html>
 
</html>
  
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                          Figure 2. The 3D structure of switch 18 docked to HtrA1 Visualized by Pymol.
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                      Figure 2. The 3D structure of switch 18 docked to HtrA1 Visualized by Pymol.
  
  
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                 Figure 3. this figure shows the results from the transcription and translation code showing the  
 
                 Figure 3. this figure shows the results from the transcription and translation code showing the  
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                      variation of mRNA and protein concentrations with time compared with the wet lab results.
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                                  variation of mRNA and protein concentrations with time.
  
  

Revision as of 17:23, 11 October 2022


HtrA1 Switch number 18

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGSGS linker (BBa_J18921), seed peptide (BBa_K4165012), GGSGGGGG linker (BBa_K4165019), seed peptide (BBa_K4165012), GSGSGS linker (BBa_J18921), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).

Usage and Biology

Switch 18 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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 379
    Illegal AgeI site found at 115
  • 1000
    COMPATIBLE WITH RFC[1000]

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. 

                    Figure 1. The 3D structure of switch 18 modeled by TRrosetta. Red: Tau binding peptides, 
                                  blue: H1A peptide, cyan: inhibitor, and green: linkers


Docking

switch 18 vs HtrA1:

ΔG = -19.516 

                      Figure 2. The 3D structure of switch 18 docked to HtrA1 Visualized by Pymol.


Mathematical modeling

Transcription rate and translation rate under T7 promotor

the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) beside with the estimated results from the wet lab. 


                Figure 3. this figure shows the results from the transcription and translation code showing the 
                                  variation of mRNA and protein concentrations with time.