Difference between revisions of "Part:BBa K4165253"

 
 
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<partinfo>BBa_K4165253 short</partinfo>
 
<partinfo>BBa_K4165253 short</partinfo>
  
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Long description
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This composite part encodes for the Dockerin module (BBa_K3396000) tagged with a GST tag (BBa_K4165070).
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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The Dockerin S. module comes from the C. thermocellum scaffoldin and it could recognize and bind tightly to its complementary counterpart Cohesin 2. 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.
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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.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4165253 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165253 SequenceAndFeatures</partinfo>
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===Dry Lab Characterization===
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<html>
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<p style=" font-weight: bold; font-size:14px;"> 1.1. Modeling </p>
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we decided to tag Docs with GST and His to increase the yield by the GST tag. we validated the effectiveness of the GST tag addition in the dry lab by modeling the protein and performing a quality assessment for the model to predict the initial stability of the Protein. Hence, the model is designed with several tools ab-intio-based tools and Template based tool (please refer to our <a href="https://2022.igem.wiki/cu-egypt/ProteinModelling.html">Modeling page)</a>.</p>.
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<p style=" font-weight: bold; font-size:14px;"> GST-Docs </p>
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/doc-gst.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                Figure 1.: Predicted 3D structure of GST-Docs designed by RosettaFold tool displayed by Pymol.
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===WetaLab Results===
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In the wet lab, we started with cloning in DH5 alpha using the pJET vector. Then we extract the plasmid by manual miniprep, then we restrict the GST-Docs gene and ligate it in pGS-21a followed by the expression in BL-21. We performed two different kinds of lysis to extract the protein to find which lysis buffer would give the better yield. We also quantified the protein expression before and after induction using BCA assay. Ultimately, we tested the GST-docs affinity by the pulldown assay against the His Coh.
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<p style=" font-weight: bold; font-size:14px;"> Ligation reaction between GST Doc with pJET cloning vector </p>
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We used T4 ligase to ligate GST Doc with pJET cloning vector, so we incubated GST Doc with pJET overnight at 15°C.
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<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ligation-of-gst-doc.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
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            Figure 2. This figure shows the ligation between GST Doc with pJET cloning vector, The band of ligation
 +
            has appeared at the appropriate size 4005, but the ligation reaction didn’t ligate all inserts with
 +
            plasmid, so there are 2 other bands representing pJET and GST-DOCs, and the ligation band appeared faint.
 +
 +
<p style=" font-weight: bold; font-size:14px;"> Transformation of GST DOC in DH-5 alpha using pJET cloning vector and in BL-21 using pGS-21a expression vector.</p>
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We transform the GST Doc in DH-5 alpha using the pJET cloning vector and in BL-21 using the pGS-21a expression vector by the TSS protocol as it shows the best transformation efficiency when compared to Calcium Chloride buffer or Calcium Chloride and Magnesium Chloride buffer. Transformation efficiency was calculated for both GST Doc in the pJET cloning vector and in the pGS-21a expression vector and was found to be =155000 transformants/μg and =170000 No. of transformants/μg, respectively.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-doc-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                                  Figure 3. The transformed plate of GST Doc + pJET.
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<p style=" font-weight: bold; font-size:14px;"> Minirprep of pJET cloning vector containing GST Doc </p>
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Miniprep is a technique used to extract the plasmid containing the gene that encodes for the protein, so we performed miniprep to extract pJET containing Gst Doc.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/miniprep-of-gst-doc.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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          Figure 4. This figure shows the miniprep of pJET cloning vector containing GST Doc, The band has appeared
 +
                                                  at the appropriate size 4005.
 +
 +
<p style=" font-weight: bold; font-size:14px;"> Transformation of GST Doc in BL-21 using pGS-21a expression vector </p>
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-doc-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
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                                  Figure 5. The transformed plate of GST Doc + pGS-21a
 +
 +
<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for GST DOC </p>
 +
To extract the protein from BL-21, we used 2 methods which are a chemical method and a physical method using sonication, and check the best result by BCA assay. The results of the 2 extraction methods show that the chemical method is more efficient, so we optimized our protocol to use chemical lysis for GST Doc.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/gst-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                    Figure 6. This graph shows a significant difference between chemical lysis and sonication for GST Doc.
 +
<p style=" font-weight: bold; font-size:14px;"> BCA assay results of protein extraction </p>
 +
BCA assay is a technique used to check the concentration of the protein, it depends on the color of the BCA working solution, which is directly proportional to the concentration of the protein concentration.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/bca-gst-doc.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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      Figure 7. This figure shows the concentration of our protein using BCA assay, which is found to be 0.828894525
 +
<p style=" font-weight: bold; font-size:14px;"> Pull-down assay of His COH with GST DOC and His DOC with GST COH </p>
 +
Pull-down assay is a one-step technique that is used to check protein-protein interaction. we performed the pull-down assay for GST Doc to check its binding against His Coh. Then we check the interaction between GST Doc against His Coh by BCA assay.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/coh-vs-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                    Figure 8. This graph illustrates that the binding between His Doc with GST Coh is more stable than that
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                                                          of His Coh with GST Doc.
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===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
 +
  
  

Latest revision as of 02:46, 14 October 2022


GST-DocS

This composite part encodes for the Dockerin module (BBa_K3396000) tagged with a GST tag (BBa_K4165070).


Usage and Biology

The Dockerin S. module comes from the C. thermocellum scaffoldin and it could recognize and bind tightly to its complementary counterpart Cohesin 2. 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

1.1. Modeling

we decided to tag Docs with GST and His to increase the yield by the GST tag. we validated the effectiveness of the GST tag addition in the dry lab by modeling the protein and performing a quality assessment for the model to predict the initial stability of the Protein. Hence, the model is designed with several tools ab-intio-based tools and Template based tool (please refer to our Modeling page).

.

GST-Docs 

                Figure 1.: Predicted 3D structure of GST-Docs designed by RosettaFold tool displayed by Pymol.

WetaLab Results

In the wet lab, we started with cloning in DH5 alpha using the pJET vector. Then we extract the plasmid by manual miniprep, then we restrict the GST-Docs gene and ligate it in pGS-21a followed by the expression in BL-21. We performed two different kinds of lysis to extract the protein to find which lysis buffer would give the better yield. We also quantified the protein expression before and after induction using BCA assay. Ultimately, we tested the GST-docs affinity by the pulldown assay against the His Coh.

Ligation reaction between GST Doc with pJET cloning vector

We used T4 ligase to ligate GST Doc with pJET cloning vector, so we incubated GST Doc with pJET overnight at 15°C. 

           Figure 2. This figure shows the ligation between GST Doc with pJET cloning vector, The band of ligation 
            has appeared at the appropriate size 4005, but the ligation reaction didn’t ligate all inserts with 
            plasmid, so there are 2 other bands representing pJET and GST-DOCs, and the ligation band appeared faint.

Transformation of GST DOC in DH-5 alpha using pJET cloning vector and in BL-21 using pGS-21a expression vector.

We transform the GST Doc in DH-5 alpha using the pJET cloning vector and in BL-21 using the pGS-21a expression vector by the TSS protocol as it shows the best transformation efficiency when compared to Calcium Chloride buffer or Calcium Chloride and Magnesium Chloride buffer. Transformation efficiency was calculated for both GST Doc in the pJET cloning vector and in the pGS-21a expression vector and was found to be =155000 transformants/μg and =170000 No. of transformants/μg, respectively. 

                                  Figure 3. The transformed plate of GST Doc + pJET.

Minirprep of pJET cloning vector containing GST Doc

Miniprep is a technique used to extract the plasmid containing the gene that encodes for the protein, so we performed miniprep to extract pJET containing Gst Doc. 

          Figure 4. This figure shows the miniprep of pJET cloning vector containing GST Doc, The band has appeared 
                                                 at the appropriate size 4005.

Transformation of GST Doc in BL-21 using pGS-21a expression vector 

                                  Figure 5. The transformed plate of GST Doc + pGS-21a

Comparison between chemical lysis and sonication for GST DOC

To extract the protein from BL-21, we used 2 methods which are a chemical method and a physical method using sonication, and check the best result by BCA assay. The results of the 2 extraction methods show that the chemical method is more efficient, so we optimized our protocol to use chemical lysis for GST Doc. 

                   Figure 6. This graph shows a significant difference between chemical lysis and sonication for GST Doc.

BCA assay results of protein extraction

BCA assay is a technique used to check the concentration of the protein, it depends on the color of the BCA working solution, which is directly proportional to the concentration of the protein concentration. 

      Figure 7. This figure shows the concentration of our protein using BCA assay, which is found to be 0.828894525

Pull-down assay of His COH with GST DOC and His DOC with GST COH

Pull-down assay is a one-step technique that is used to check protein-protein interaction. we performed the pull-down assay for GST Doc to check its binding against His Coh. Then we check the interaction between GST Doc against His Coh by BCA assay. 

                    Figure 8. This graph illustrates that the binding between His Doc with GST Coh is more stable than that 
                                                          of His Coh with GST Doc.

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