Coding

Part:BBa_K4165255

Designed by: Esraa Elmligy   Group: iGEM22_CU_Egypt   (2022-10-08)
Revision as of 14:26, 12 October 2022 by Ahmedsameh (Talk | contribs)


GST-Coh2-G4S- TD28REV

This part consists of the Cohesin 2 module (BBa_K4165003) connected to the tau binding peptide TD28REV (BBa_K4165006) through a flexible linker of GGGGS (BBa_K4165068) repeated 3 times and tagged with a GST tag (BBa_K4165070).

Usage and Biology

The part is considered an integral part of the snitch system in which it directs the Trim21 (E3) ligase to the tau protein in order to force the degradation of tau proteins which causes Alzheimer's Diseases via the proteasomal degradation pathway.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 689
    Illegal SapI.rc site found at 85

Dry-Lab Characterization

Modelling 

                  Figure 1. A 3D model showing the structure of GST-CoH2-Linker-TD28REV (Model 3 - TRrosetta)

WetLab Results

Transformation of GST COH (L) TD21 Rev in DH-5 alpha using pJET cloning vector 

                               Figure 2. Transformed plate of GST COH (L) TD28 Rev + pJET

Transformation of GST COH (L) TD28 Rev in BL-21 using pGS-21a expression vector 

                             Figure 3. Transformed plate of GST COH (L) TD28 Rev + pGS-21a

Comparison between chemical lysis and sonication for GST COH TD28 Rev 

                      Figure 4. this graph shows a difference between chemical lysis and sonication for GST COH TD28 
                       Rev, after we had the results we optimized our protocol to use chemical lysis for GST COH (L) 
                                                                TD28Rev.

SDS PAGE of induced and non induced samples of GST COH TD28 Rev 

               Figure 5. This figure shows the comparison between the induced and non induced samples of GST COH 
               TD28Rev, where well no.2 is the induced sample of GST COH TD28 Rev while well no.6 is the non induced 
               sample of GST COH TD28Rev showing that our protein is induced effectively owing to our right choice of 
                      I                       PTG, time interval and concentration

Pull down assay His Trim (L) DOC against GST COH WWW and GST COH TD28Rev 

               Figure 6. This graph shows the comparison of pull down assay between His Trim21 (L) DOC with GST COH 
               WWW and GST COH TD28Rev showing that the interaction between His Trim21 (L) DOC and GST COH WWW is 
               better than that of His Trim21 (L) DOC with GAT COH TD28Rev as the concentration of elution of His 
                  Trim21 (L) DOC with GST COH WWW is more than that of His trim21 (L) DOC with GST COH TD28Rev

Pull down assay of Tau aggregates against GST COH WWW and GST COH TD28Rev 

               Figure 7. This graph shows the comparison of pull down assay between Tau aggregates with GST COH WWW and 
               Tau aggregates with GST COH TD28Rev, showing that the interaction between Tau aggregates with GST COH 
                WWW is better than that of Tau aggregates with GST COH TD28rev as the concentration of elution of Tau 
                aggregates with GST COH WWW is more than that of Tau aggregates with GST COH TD28Rev, illustrating that 
                           WWW could be a better candidate for binding to the Tau aggregates



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