Difference between revisions of "Part:BBa K4165254"
Line 12: Line 12:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4165254 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165254 SequenceAndFeatures</partinfo>
 +
 +
===Dry lab characterization===
 +
<p style=" font-weight: bold; font-size:14px;"> Modeling </p>
 +
Coh2 was modeled tagged with GST to purify it and measure its expression yield, the models were done using (Alphafold - Modeller - trRosetta - Rosettafold) and the top models were obtained from Alphafold and trRosetta ranking 5 out of 6 according to our QA code.
 +
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/gst-coh.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
 +
          Figure 1.: Predicted 3D structure of Coh2 protein tagged by GST designed by AlphaFold tool visualized on Pymol.
 +
 +
<p style=" font-weight: bold; font-size:13px;"> Table 1: Quality assessment parameters of GST-Coh2 model. </p>
 +
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/gst-cohqa.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
 +
<p style=" font-weight: bold; font-size:14px;"> Docking </p>
 +
GST-Coh2 is docked to His-DocS
 +
<p style=" font-weight: bold; color: red; font-size:14px;"> ΔG = -13.15 kcal/mol</p>
 +
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/switches/gst-coh2-galaxy.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
 +
          Figure 2.: 3D structure of GST-Coh2 docked with His-DocS on Galaxy and visualized on pymol.
  
 
===WetLab Results===
 
===WetLab Results===
Line 18: Line 44:
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-coh.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-coh.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
−
                                           Figure 1. Transformed plate of GST COH + pGS-21a  
+
                                           Figure 3. Transformed plate of GST COH + pGS-21a  
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of GST COH in DH-5 alpha using pJET vector </p>
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of GST COH in DH-5 alpha using pJET vector </p>
 
<html>
 
<html>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-coh-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-coh-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
−
                                         Figure 2. Transformed plate of GST COH + pJET  
+
                                         Figure 4. Transformed plate of GST COH + pJET  
 
<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for GST COH </p>
 
<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for GST COH </p>
 
<html>
 
<html>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/gst-coh2.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/gst-coh2.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
−
Figure 3. This graph shows a highly significant difference between the chemical lysis and sonication for GST COH, after we had this result we optimized our protocol to use sonication for GST COH  
+
Figure 5. This graph shows a highly significant difference between the chemical lysis and sonication for GST COH, after we had this result we optimized our protocol to use sonication for GST COH  
  
 
===References===
 
===References===

Revision as of 09:52, 12 October 2022


GST-Coh2

This part encodes Cohesin 2 protein tagged with GST for its purification and characterization

Usage and Biology

The Cohesin 2 module comes from the C. thermocellum scaffoldin and it could recognize and bind tightly to its complementary counterpart Dockerin S. The Coh2–DocS pair represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities, ranging from one of the highest known affinity constants between two proteins to relatively low-affinity interactions. This serves an essential role in the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome), this interaction happens in two different forms, called the dual binding mode, in a calcium-dependent manner due to the presence of a calcium-binding site in the dockerin protein.

We used the DocS-Coh2 binding in our Snitch system to form the PROTAC pair that will conjugate E3 ligase trim 21 (BBa_K4165001) with the binding peptide for our targeted protein tau. 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 SapI.rc site found at 85

Dry lab characterization

Modeling

Coh2 was modeled tagged with GST to purify it and measure its expression yield, the models were done using (Alphafold - Modeller - trRosetta - Rosettafold) and the top models were obtained from Alphafold and trRosetta ranking 5 out of 6 according to our QA code. 

          Figure 1.: Predicted 3D structure of Coh2 protein tagged by GST designed by AlphaFold tool visualized on Pymol.

Table 1: Quality assessment parameters of GST-Coh2 model.

Docking

GST-Coh2 is docked to His-DocS

ΔG = -13.15 kcal/mol

          Figure 2.: 3D structure of GST-Coh2 docked with His-DocS on Galaxy and visualized on pymol.

WetLab Results

Transfotmation of GST COH in BL-21 using pGS-21a vector 

                                          Figure 3. Transformed plate of GST COH + pGS-21a

Transformation of GST COH in DH-5 alpha using pJET vector 

                                        Figure 4. Transformed plate of GST COH + pJET

Comparison between chemical lysis and sonication for GST COH

Figure 5. This graph shows a highly significant difference between the chemical lysis and sonication for GST COH, after we had this result we optimized our protocol to use sonication for GST COH

References

1. Brás, J. L., Carvalho, A. L., Viegas, A., Najmudin, S., Alves, V. D., Prates, J. A., Ferreira, L. M., Romão, M. J., Gilbert, H. J., & Fontes, C. M. (2012). Escherichia coli Expression, Purification, Crystallization, and Structure Determination of Bacterial Cohesin–Dockerin Complexes. Methods in Enzymology, 510, 395-415. https://doi.org/10.1016/B978-0-12-415931-0.00021-5

2. Slutzki, M., Ruimy, V., Morag, E., Barak, Y., Haimovitz, R., Lamed, R., & Bayer, E. A. (2012). High-Throughput Screening of Cohesin Mutant Libraries on Cellulose Microarrays. Methods in Enzymology, 510, 453-463. https://doi.org/10.1016/B978-0-12-415931-0.00024-0

3. Stahl, S. W., Nash, M. A., Fried, D. B., Slutzki, M., Barak, Y., Bayer, E. A., & Gaub, H. E. (2012). Single-molecule dissection of the high-affinity cohesin–dockerin complex. Proceedings of the National Academy of Sciences, 109(50), 20431-20436.

4. Karpol A, Kantorovich L, Demishtein A, Barak Y, Morag E, Lamed R, Bayer EA. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. J Mol Recognit. 2009 Mar-Apr;22(2):91-8. doi: 10.1002/jmr.926. PMID: 18979459.

5. Wojciechowski, M., Różycki, B., Huy, P.D.Q. et al. Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module. Sci Rep 8, 5051 (2018). https://doi.org/10.1038/s41598-018-23380-9