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Team:Goettingen/project overview/project wetlab
From 2014.igem.org
Project
Result: Wetlab
Approach
Our experimental approach can be summarized as follows:
1. Organisms and surface proteins selection
2. Surface protein amplification and cloning
3. Yeast two-hybrid assay with an existing peptide library
4. Peptide functionalization
Results summary
These are the genes and the organisms we selected:
| Gene | Organism |
|---|---|
| mp65 | C. albicans |
| tos1 | C. albicans |
| sim1 | C. albicans |
| als1 | C. albicans |
| als3 | C. albicans |
| ssr1 | C. glabrata |
| pir4 | C. glabrata |
| scw4 | C. glabrata |
| pir3 | C. glabrata |
| utr2 | C. glabrata |
| crf2full | A. fumigatus |
| crf2act | A. fumigatus |
| bgleno | A. fumigatus |
| ecm33 | A. fumigatus |
| sun1 | A. fumigatus |
| roda | A. fumigatus |
| bgleex | A. fumigatus |
| prm1 b | A. fumigatus |
| crf1 | A. fumigatus |
| roda | A. nidulans |
| xlna | A. nidulans |
| npc2 | A. nidulans |
| sho1 | A. nidulans |
| eglc | A. nidulans |
| eglc | A. fumigatus |
| CWP1 | S. cerevisiae |
| CWP2 | S. cerevisiae |
| TIR4 | S. cerevisiae |
| TIR1 | S. cerevisiae |
| MID2 | S. cerevisiae |
| SED1 | S. cerevisiae |
| ama1 | T. gondii |
| rom5 | T. gondii |
| rom4 | T. gondii |
| gra7 | T. gondii |
The results for all the genes we selected are summarized in this file.
Result: Dry lab
Homology modelling
We modeled the 3D structure of our peptide-containing scaffolds (domain B1 of protein G from Staphylococcus aureus) by homology modelling. We used the Modeller library to accomplish that; since the peptide is about 20-25 amino acids long, it can be modeled as a loop inside the B1 domain scaffold for which there are already a number of 3D structures available at the Protein Data Bank.
This is the general homology modeling procedure we followed. All peptides were cloned inside a scaffold (B1 domain of protein G from Staphylococcus aureus. The peptides were short enough to be modeled as an internal loop by loop refinement; these structures should only be considered as a working model, since we do not have direct crystallographic data. A detailed procedure can be found in this link.
Results summary
Protein G scaffold
The following table is a summary of the models we generated for our peptides inside the protein G scaffold. Their main purpose is to give us an idea if the calculated energy profile matches our assumption that the peptide is being exposed to the exterior of the scaffold. These models are in no way definitive, since we lack direct crystalographic data.
| Peptide | Video | PDB | Peptide location | QMEAN score | Overall DOPE score | Energy profile |
|---|---|---|---|---|---|---|
| Prey 3 | Download | K17-E42 | 0.512 | -7053.966797 | Download | |
| Prey 4.1 | Download | K17-E44 | 0.468 | -7246.956055 | Download | |
| Prey 5 | Download | K17-E44 | 0.495 | -7501.071777 | Download | |
| Prey 13 | Download | K17-E42 | 0.535 | -7729.056152 | Download | |
| Prey 15 | Download | K17-E42 | 0.441 | -7685.998535 | Download |
*The peptide is shown in red and the scaffold in blue.
GFP scaffold
These are the structures we generated for our peptides inside a GFP scaffold.
| Peptide | Video | PDB | Peptide location | QMEAN score | Overall DOPE score | Energy profile |
|---|---|---|---|---|---|---|
| Prey 3 |  |
Download | E103-E127 | 0.43 | -26798.28125 | Download |
| Prey 4.1 |  |
Download | E103-E127 | 0.448 | -27891.212891 | Download |
| Prey 5 |  |
Download | E103-E127 | 0.483 | -26633.4375 | Download |
| Prey 13 |  |
Download | E103-E127 | 0.404 | -27075.160156 | Download |
| Prey 15 |  |
Download | E103-E127 | 0.413 | -26713.355469 | Download |
*The peptide is shown in red and the scaffold in green.
References
1. N. Eswar, M. A. Marti-Renom, B. Webb, M. S. Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali. Comparative Protein Structure Modeling With MODELLER. Current Protocols in Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30, 2006.
2. M.A. Marti-Renom, A. Stuart, A. Fiser, R. Sánchez, F. Melo, A. Sali. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29, 291-325, 2000.
3. A. Sali & T.L. Blundell. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815, 1993.
4. A. Fiser, R.K. Do, & A. Sali. Modeling of loops in protein structures, Protein Science 9. 1753-1773, 2000.
