Composite

Part:BBa_K4165202

Designed by: Mennatallah Mahmoud Mohamed Abdelzaher Turky   Group: iGEM22_CU_Egypt   (2022-10-01)
Revision as of 13:38, 12 October 2022 by Engy99 (Talk | contribs) (→‎Usage and Biology)


Trim-(GGGGS)3-Docs

This parts code for the trim21 E3 ligase having his PRYSPRY domain truncated (BBa_K3396007), fused to type 1 Dockerin module derived from Clostridium thermocellum cellulosome scaffoldin using Glycine serine flexible linker repeated three times to maintain part flexibility needed during target ubiquitination.

Usage and Biology

this part is the main part in our Trim System (Snitch System) , it is supposed to bind to the protac (Coh2-linker-tau_binding_peptide) which in turn will bind to tau protein. The hypothesis is when the binding occurs between the three parts, tTrim21 will recruit E2 conjugating enzyme that carries ubiquitin and move the ubiquitin from the E2 to tau. After ubiquitination, tau protein is supposed to be degraded by 26S proteasome.

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.rc site found at 535

Modeling

tTrim21-(G4S)3-DocS is modeled by AlphaFold2, ITASSER, MODELLER, Robetta and TrRosetta, best model obtained from TrRosetta. 

                   Figure 1.: Predicted 3D structure of our fusion protein tTrim21-(G4S)3-DocS.


Table 1: Quality assessment parameters of tTrim21-(G4S)3-DocS. model.



WetLab Results

Transformation of His Trim21 (L) Doc in BL-21 using pGS-21a 

                             Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a

Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector 

                              Figure 3. Transformed plate of His Trim21 (L) Doc + pJET

Comparison between chemical lysis and sonication for His Trim21 (L) DOC 

                      Figure 4. This graph shows a significant difference between chemical lysis and sonication
                            for His Trim21 (L) DOC, after we had the results, we optimized our protocol to
                                         use sonication for His Trim21 (L) DOC

SDS PAGE of induced and non-induced samples of His Trim 21 (L) DOC 

             Figure 5. This figure shows the comparison between the induced and non-induced samples of His Trim21 
              (L) DOC, where well no.1 is the non-induced sample while well no.3 is the induced sample showing that 
              our protein is induced effectively owing to our right choice of IPTG, time interval and concentration

Pull down assay of His Trim21 (L) DOC against GST COH WWW and GST COH TD28 Rev 

             Figure 6. This graph shows the comparison of pull down assay between His Trim (L) DOC against GST COH WWW and 
              GST COH TD28 Rev, showing that the interaction between His Trim21 (L) DOC and GST COH WWW is better than that 
              of His Trim (L) DOC and GST COH TD28 Rev as the concentration of the elution coming from the pull down assay 
              of His Trim21 (L) DOC and GST COH WWW is more than that of His Trim21 (L) DOC and GST COH TD28rev


References

1-Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.


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