Composite

Part:BBa_K4165035

Designed by: Salma Sobhy Sadek   Group: iGEM22_CU_Egypt   (2022-09-30)
Revision as of 15:32, 12 October 2022 by Engy99 (Talk | contribs) (Usage and Biology)


HtrA1 Switch number 15

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), TD28rev (BBa_K4165006), GGSGGGGG linker (BBa_K4165019), WWW (BBa_K4165007), GSGSGS linker (BBa_J18921), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).

Usage and Biology

Switch 15 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.

The TD28REV and WWW peptides which are 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 which is the inhibitor which is mainly a serine protease inhibitor, and since our protease is a serine protease, so it will act and inhibit the Protein. The whole construction was similarly proved from literature.

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]

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 15 is modeled by TRrosetta. Red: Tau binding peptides, 
                                      blue: H1A peptide, cyan: inhibitor and green: linkers


Docking

switch 15 vs HtrA1 trimer:

ΔG = -15.52

                             Figure 2. The 3D structure of switch 15 docked to HtrA1 displayed on Pymol.

Mathematical modeling

Transcription rate and translation rate under T7 promoter

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.


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