Difference between revisions of "Part:BBa K3396000"

Revision as of 22:38, 11 October 2022

DocS

The Coch2 module binds DocS (BBa_K3396000) modules constitutively.

Usage and Biology

The DocS[1] module comes from The C. thermocellum scaffoldin and it could recognize and bind tightly to complementary Coh2 modules. 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.

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
COMPATIBLE WITH RFC[1000]

Improvement by CU_Egypt team 2022

From literature we found that the yield of expression of Docs is very low so we decided to test it with different tags GST and His to see their effects on its stability and expression yield. Also, we optimized the sequence to be expressed in E-coli. in addition, there is no characterization for Docs on the registry so we expressed and characterized it by different methods such as Agarose gel electrophoresis, SDS PAGE, transformation efficiency, affinity chromatography, and Bradford assay. it's proved by wet lab results that the expression yield of Docs has raised with tagging by GST.

1. Dry Lab

1.1. Modeling

Docs has been tagged with GST and His for purification and increasing the yield by the GST tag. then the model designed by several tools to get the best model.

GST-Docs

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

                                Table 1.: QA scores by SWISS model tool of GST-Docs structure.

His-Docs

                       Figure 2.: Predicted 3D structure of His-Docs designed by TRrosetta tool displayed by Pymol.

                              Table 2.: QA scores by SWISS model tool of His-Docs structure.

1.2. Docking

Tagged Docs with GST and His has been docked with Coh2 tagged with GST and His by different tools to get the best model. Best docked structures were retrieved from ClusPro and Galaxy webservers according to our ranking code.

GST-Docs VS His-Coh by Cluspro

                       Figure 3.: Docked structure of GST-Docs VS His-Coh2 designed by Cluspro displayed by Pymol.

GST-Docs VS His-Coh2 by Galaxy

                         Figure 4.: Docked structure of GST-Docs VS His-Coh2 designed by Galaxy visualized by Pymol.

His-Docs VS GST-Coh by ClusPro

                          Figure 5.: Docked structure of His-Docs VS GST-Coh2 designed by ClusPro visualized by Pymol.

His-Docs VS GST-Coh2 by Galaxy

                       Figure 6.: Docked structure of His-Docs VS GST-Coh2 designed by Galaxy visualized by Pymol.

Binding energies of Docs VS Coh2

             Table 3.: Binding energies of Docs VS Coh2 tagged with GST and His designed by Galaxy and ClusPro.

All of docking results were ranked using our code for calculating the binding affinity.

1.3. Additional Dry Lab characterization

pI: 5.30

M.Wt.: 16751.19 Da

WetLab Results

Transformation of His Doc in BL-21 using pGS-21a vector

                                       Figure 7. Transformed plate of His Doc + pGS-21a

Transformation of His Doc in DH-5 alpha using pJET vector

                                       Figure 8. Transformed plate of His Doc + pJET

Comparison between chemical lysis and sonication for His DOC

              Figure 9. This graph shows the difference between chemical lysis and sonication for His DOC,
                    after we had the results, we optimized our protocol to use chemical lysis for His DOC

SDS PAGE for induced and non induced samples of His DOC

Figure 10. This figure shows the comparison between induced and non induced samples of His DOC where well no.2 is the non induced sample while well no.4 is the induced sample showing that our protein is induced effectively owing to our right choice of IPTG, time interval and concentration

Reference

[1] BARAK Y, HANDELSMAN T, NAKAR D, et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction [J]. J Mol Recognit, 2005, 18(6): 491-501.

[2] Kazutaka Sakka, Yuka Sugihara, Sadanari Jindou, Makiko Sakka, Minoru Inagaki, Kazuo Sakka, Tetsuya Kimura, Analysis of cohesin–dockerin interactions using mutant dockerin proteins, FEMS Microbiology Letters, Volume 314, Issue 1, January 2011, Pages 75–80, https://doi.org/10.1111/j.1574-6968.2010.02146.x

[3] Lawrie, J., Song, X., Niu, W., & Guo, J. (2018). A high throughput approach for the generation of orthogonally interacting protein pairs. Scientific Reports, 8. https://doi.org/10.1038/s41598-018-19281-6

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                 Figure 9. This graph shows the difference between chemical lysis and sonication for His DOC,
                       after we had the results, we optimized our protocol to use chemical lysis for His DOC
+<p style=" font-weight: bold; font-size:14px;"> SDS PAGE for induced and non induced samples of His DOC </p>
+<html>
+<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/sds-of-his-doc.jpg" style="margin-left:200px;" alt="" width="500" /></p>
+</html>
+Figure 10. This figure shows the comparison between induced and non induced samples of His DOC where well no.2 is the non induced sample while well no.4 is the induced sample showing that our protein is induced effectively owing to our right choice of IPTG, time interval and concentration
 ===Reference===
 [1] BARAK Y, HANDELSMAN T, NAKAR D, et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction [J]. J Mol Recognit, 2005, 18(6): 491-501.