Team:INSA-Lyon/Molecular

From 2014.igem.org

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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/2/2b/CurlyonFoldedHis1.jpg" alt="floating His1" width="300px"/></div></td>
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                             <div align = "justify"><p>
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The results of the wet lab however, showed <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu1">no particular decrease</a> in curli formation between tagged and wildtype-producing <i>E.coli</i>, and added to this, the nickel chelation seemed greater with the tags than without it, although the difference was hard to catch since <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">CsgA wildtype is already able to chelate nickel </a>. This means that <b>the floating conformation seems more likely to happen <i>in vivo</i> than the folded one</b>.
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The results of the wet lab however, showed <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu1">no particular decrease</a> in curli formation between tagged and wildtype-producing <i>E.coli</i>. Though we do not know the extent to which the folded conformation may hinder polymerisation, we may conclude that <!--, and added to this, the nickel chelation seemed greater with the tags than without it, although the difference was hard to catch since <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">CsgA wildtype is already able to chelate nickel </a>. This means that--> <b>the floating conformation seems more likely to happen <i>in vivo</i> than the folded one</b>.
                             </p></div>
                             </p></div>

Revision as of 01:38, 18 October 2014

Curly'on - IGEM 2014 INSA-LYON

One of the main goals of our modeling work this year was to understand the structure of the curlin subunit protein, CsgA and its behavior when engineered with a tag constituted of either six histidines (that we will call His1-tag from now on) or twice that motif (His2-tag), since this peptide is known for its nickel chelation properties.
We then discussed over our results with the wet lab members to define a way to confirm the accuracy of our model, and so we were able to assess that, in accordance with literature, the best position for the tag was by the C-terminus end of the protein. We also determined that the His-tag was more likely to take a floating conformation instead of folding itself around CsgA.


  • Methods


  • CsgA Engineering


  • Ni-Chelation




Conclusion

Overall sum up

Through our molecular study of a CsgA protein engineered with either His1-tag or His2-tag, we came to the conclusion that since it has a longer reach and its mobility makes it more available for chelation, using a tag positioned by the C-terminus of the protein is more relevant than placing it in the middle of the sequence, although doing so may provide a little more chelation power as long as the tag isn't too long.
We also showed that there are two possible conformations : one is folded on the side of CsgA and a priori does not increase the already-existing chelating power of CsgA; the other is a "floating" conformation where the tag is not attracted to the protein and is able to improve its chelating power by up to 25%!

What we couldn't achieve

Unfortunately, having very little time and people, there are a few things we couldn't investigate as extensively as we wanted. Here are a few of those things:

  • more simulations with His2-tag. Since they took an awful lot of time, we only ran a handful of them;
  • modelisation of the docking between two CsgA proteins, and the influence of the His-tags, that our lack of experience prevented us from conducting;
  • find out just how many tags can be added without altering the protein properties of adherence and polymerization;