Team:Goettingen/project overview/project wetlab

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<h2 id="wlresults">Results summary</h2>
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<p>These are the genes and the organisms we selected:</p><br />
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<p>We chose the five fungi Aspergillus fumigatus, A. nidulans, Candida glabrata, C. albicans and Saccharomyces cerevisiae as well as the parasite Toxoplasma gondii for our project. In the first step, we amplified all in all 35 different genes encoding surface proteins of the mentioned organisms in a standard PCR reaction (compare table 1). We were able to transform 20 of our 35 genes into a so called bait vector (contains a DNA-binding domain) and performed the Yeast-Two-Hybrid (Y2H) screening with them. Thereby, we accomplished to find more than 3000 probable interaction partners for our surface proteins. </p><br /><br />
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<p><b>Table 1| Selected surface proteins and the corresponding organism.</b></p></br></br>
<center><table class="table1" style="width:700px">
<center><table class="table1" style="width:700px">

Revision as of 19:38, 17 October 2014

Result: Wetlab

Approach

Our experimental approach can be summarized as follows:


  1. 1. Organisms and surface proteins selection

  2. 2. Surface protein amplification and cloning

  3. 3. Yeast two-hybrid assay with an existing peptide library

  4. 4. Peptide functionalization





Results summary

We chose the five fungi Aspergillus fumigatus, A. nidulans, Candida glabrata, C. albicans and Saccharomyces cerevisiae as well as the parasite Toxoplasma gondii for our project. In the first step, we amplified all in all 35 different genes encoding surface proteins of the mentioned organisms in a standard PCR reaction (compare table 1). We were able to transform 20 of our 35 genes into a so called bait vector (contains a DNA-binding domain) and performed the Yeast-Two-Hybrid (Y2H) screening with them. Thereby, we accomplished to find more than 3000 probable interaction partners for our surface proteins.



Table 1| Selected surface proteins and the corresponding organism.



Gene Organism
mp65C. albicans
tos1C. albicans
sim1C. albicans
als1C. albicans
als3C. albicans
ssr1C. glabrata
pir4C. glabrata
scw4C. glabrata
pir3C. glabrata
utr2C. glabrata
crf2fullA. fumigatus
crf2actA. fumigatus
bglenoA. fumigatus
ecm33A. fumigatus
sun1A. fumigatus
rodaA. fumigatus
bgleexA. fumigatus
prm1 bA. fumigatus
crf1A. fumigatus
rodaA. nidulans
xlnaA. nidulans
npc2A. nidulans
sho1A. nidulans
eglcA. nidulans
eglcA. fumigatus
CWP1S. cerevisiae
CWP2S. cerevisiae
TIR4S. cerevisiae
TIR1S. cerevisiae
MID2S. cerevisiae
SED1S. cerevisiae
ama1T. gondii
rom5T. gondii
rom4T. gondii
gra7T. 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. 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. 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. 3. A. Sali & T.L. Blundell. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815, 1993.


  4. 4. A. Fiser, R.K. Do, & A. Sali. Modeling of loops in protein structures, Protein Science 9. 1753-1773, 2000.