Part:BBa_K2607000:Design
DiphTox (DT)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Our team performed MUSTANG server alignment test to see whether there is significant difference of 3D structure between DiphTox (DT) and original diphtheria toxin. The result of RMSD (Root Mean Square Deviation) between DT and original toxin is 0.969 Å. For the result of RMSD is below the value of 2 Å, it means there is no significant structural difference between these two molecules.
Characterization or purification of those proteins would promote the usage of His-tag; therefore, insertion of His-tag inside the sequence is essential. To ensure the slightest change of tertiary structures of each protein, we would need to find out the secondary structure and surface accesibility via NetSurfP analyser version 1.1 (http://www.cbs.dtu.dk/services/NetSurfP/). We would insert His-tag sequence in either no available specific protein domain or the coiled secondary structure of protein to minimize any interruptions. Table 1 shows DT data from NetSurfP server. Result from the NetSurf server, we choose C-terminus side, because it most likely turns/coils around (indicated by has high number on the fourth column is closest to 1), and it is freely exposed (indicated by most left column has E alphabet).
| Class Assignment | Amino Acid | Amino Acid Number | Probability for Coil |
|---|---|---|---|
| B | I | 54 | 0.223 |
| E | K | 55 | 0.669 |
| E | S | 56 | 0.994 |
For iGEM BioBrick submission, we would use prefix and suffix that contain EcoRI and PstI restriction site. HindIII and BamHI restriction site are inserted upstream from prefix and downstream from suffix respectively. The DT contains only the last 54 amino acid from the R domain which has no cytotoxicity and significant enough to bind with HB-EGF receptor. At the downstream of the sequence, designing six histidine amino acids is essential for His-tag protein purification, as well as characterizing the DT and HB-EGF/Tar (BBa_K2607001) receptor kinetics. Ribosome binding site (RBS) is located upstream within the gBlocks containing Shine-Dalgarno sequence as follow.
Source
The native protein was taken from Corynebacterium diphtheriae. We made some modifications to render it nontoxic so it is relatively safe to conduct experiment with this engineered toxin.
References
[1]MUSTANG: A multiple structural alignment algorithm. Konagurthu AS, Whisstock JC, Stuckey PJ, Lesk AM (2006) Proteins 64, 559-574.
[2]Bordogna A, Pandini A, Bonati, L. 2010. Predicting the accuracy of protein-ligand docking on homology models. Journal of Computational Chemistry 32, 81–98.
[3]Carugo O. 2003. How root-mean-square distance (r.m.s.d.) values depend on the resolution of protein structures that are compared. Journal of Applied Crystallography 36, 125–128.