Difference between revisions of "Part:BBa K4165202"

Revision as of 22:53, 11 October 2022

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 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

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.

@@ Line 9: / Line 9: @@
 this part is the main part in our 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.
-===Source===
+===<span class='h3bb'>Sequence and Features</span>===
-Synthetic Fusion protein
-===<span class='h3bb'>Sequence and Features</span>===
-sequence was optimized for <i>E.coli</i> expression
 <partinfo>BBa_K4165202 SequenceAndFeatures</partinfo>
-===Dry Lab===
+===Modeling===
-<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
 tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS is modeled by AlphaFold2, ITASSER, MODELLER, Robetta and TrRosetta, best model obtained from TrRosetta.
 <html>
-<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/trim-g4s3-docs.png" style="margin-left:150px;" alt="" width="500" /></p>
+<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/trim-g4s3-docs.png" style="margin-left:200px;" alt="" width="500" /></p>
 </html>
-                             Figure 1.: Predicted 3D structure of our fusion protein tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS.
+                    Figure 1.: Predicted 3D structure of our fusion protein tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS.
@@ Line 38: / Line 29: @@
 /></p>
 </html>
@@ Line 46: / Line 36: @@
 <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/trim-doc-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 </html>
-                                    Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a
+                              Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a
 <p style=" font-weight: bold; font-size:14px;"> Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector </p>
 <html>
 <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-trim-doc-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 </html>
-                                    Figure 3. Transformed plate of His Trim21 (L) Doc + pJET
+                               Figure 3. Transformed plate of His Trim21 (L) Doc + pJET
 <p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for His Trim21 (L) DOC </p>
 <html>
 <p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/trim-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 </html>
-                                    Figure 4. This graph shows a significant difference between chemical lysis and sonication
+                       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
+                             for His Trim21 (L) DOC, after we had the results, we optimized our protocol to
-                                              use sonication for His Trim21 (L) DOC
+                                          use sonication for His Trim21 (L) DOC
-<p style=" font-weight: bold; font-size:14px;"> SDS PAGE of induced and non induced samples of His Trim 21 (L) DOC </p>
+<p style=" font-weight: bold; font-size:14px;"> SDS PAGE of induced and non-induced samples of His Trim 21 (L) DOC </p>
 <html>
 <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/sds-of-trim21-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 </html>
-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
+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