Team:EPF-Lausanne/Our Project/TetR mutants/Conclusion

From 2011.igem.org

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{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Conclusion}}
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!| Affinity compared to WT - in vivo
!| Affinity compared to WT - in vivo
!| Affinity compared to WT - MITOMI
!| Affinity compared to WT - MITOMI
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!| ATC repression
 
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| <html> <a href="http://partsregistry.org/Part:BBa_K613013"> BBa_K613013</a> </html>
| <html> <a href="http://partsregistry.org/Part:BBa_K613013"> BBa_K613013</a> </html>
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| E37A W43S T141A
| E37A W43S T141A
| <html> <a href="http://partsregistry.org/Part:BBa_K613015"> BBa_K613015</a> </html>
| <html> <a href="http://partsregistry.org/Part:BBa_K613015"> BBa_K613015</a> </html>
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| <html> <a href="http://partsregistry.org/Part:BBa_K613016"> BBa_K613016</a> </html>
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The '''P39K mutant''' shows in both cases (''in vivo'' and ''in vitro'') no affinity to the Ptet consensus sequence, showing that our results are consistent between the two characterizations.
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The '''V36F mutant''' either shows a stronger or a similar binding affinity compared to the WT. The differences not being striking, our characterization methods are here again consistent, with the ''in vitro'' technique being more sensitive than the ''in vivo''.
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Finally, the '''E37A W43S T141A triple mutant''' was shown ''in vitro'' to have a different consensus sequence that the wild-type Ptet sequence. However, the ''in vivo'' experiment only tested the mutant with the wild-type Ptet and shows an affinity in the range of the wild-type TetR. These two results indicate that, althoud a change in specificity, there is still crosstalk with the original Ptet sequence. This mutant is the most promising of the three characterized in our two systems, harbouring a change in specificity as well as DNA binding properties. Still, this is not an orthogonal mutant; more TetRs need to be tested before fully eliminating crosstalk with wild-type Ptet.
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The results coming from the ''in vivo'' and ''in vitro'' part are consistently related. We have tested only 3 mutants in both systems ; we would need more characterization results to improve our selection systems, especially the ''in vivo'' part.
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The MITOMI results can be found [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data here]. The in vivo characterizations are on [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Reporter_Systems/tetR#TetR_mutant_characterization this page].
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 19:39, 26 October 2011

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