Composite

Part:BBa_K5036050

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-21)
Revision as of 17:07, 25 September 2024 by Alisalah (Talk | contribs) (→‎Software Characterization)


MMP9 Nanobody3- NSP3A

Part Description

Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to NSP3 protein, which is attached to the cap from the other end.

Usage

Our TID device contains NSP3 protein linked to specific sensors (nanobodies) that detect MMP9, a protein elevated within cells after tissue injury. TID switches from off state to on state when MMP9 is present in the cell cytoplasm. This triggers the circularization of YAP mRNA, which is essential for protein production

this figure illustrates the structure of NSP3-MMP9 Nanobody which is attached to the cap of our switch .

Dry lab Characterization

We measured the effect of the nanobodies on the NSP3-CAP binding stability, so we measured NSP3-CAP before and after binding to nanobody1 and nanobody3

NSP3-CAP

this figure illustrates The estimated binding stability (ΔG) between NSP3 and the Cap binding protein at the 5’ end of the mRNA equals -13.8 kcal mol-1 .


NSP3-Cap-NB1

this figure illustrates The estimated binding stability (ΔG) between NSP3 and the Cap binding protein, in the presence of nanobody1 , at the 5’ end of the mRNA equals -27.8 kcal mol-1. .

NSP3-Cap-NB3

this figure illustrates The estimated binding stability (ΔG) between NSP3 and the Cap binding protein, in the presence of nanobody 3 , at the 5’ end of the mRNA equals -34.9 kcal mol-1. .


Then we compared between the previous three states of the 5’ prime end and we conclude that Nanobodies presence stabilized the proteins at the 5’ end.

this figure shows that adding the NB1 to the NSP3 and the Cap increased their binding stability (ΔG) from -13.8 kcal mol-1 to -27.8 kcal mol-1. While adding the NB3 intensified their binding stability (ΔG) from -13.8 kcal mol-1 to -34.9 kcal mol-1 Therefore, we used the NB3 at the 5’ end to increase its stability, and NB1 at the 3’ prime end putting its high binding stability with MMP9 in our consideration .


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1730
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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