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

From 2011.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Mutant TetRs}}
{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Mutant TetRs}}
 +
=== Strategy ===
-
We used two distinct strategies to make the mutants:  
+
Having done a litterature search, we decided to focus first on mutating some key amino acids rather than going for random mutations. The most important residues seem to be the V36, E37, P39 and Y42 ones.
 +
 
 +
 
 +
We used two distinct strategies to create these mutants:  
* <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/Site-specific_mutagenesis"> Site-specific mutagenesis </a></html>, which consists of both a plasmid amplification step with forward and reverse primers containing the targeted mutation and a selection step (enzymatic digestion of the methylated dsDNA)  
* <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/Site-specific_mutagenesis"> Site-specific mutagenesis </a></html>, which consists of both a plasmid amplification step with forward and reverse primers containing the targeted mutation and a selection step (enzymatic digestion of the methylated dsDNA)  
Line 9: Line 13:
The plasmid that we used had a C-terminal eGFP fusion to the TetR, while the linear template had a C-terminal His-tag. The promoters added were SP6 in the first case and T7 in the second.
The plasmid that we used had a C-terminal eGFP fusion to the TetR, while the linear template had a C-terminal His-tag. The promoters added were SP6 in the first case and T7 in the second.
 +
 +
 +
=== Mutants obtained ===
 +
 +
Here is a list of all the mutants we created, their characterization statut and a link to their respective PartsRegistry page.
{|  style="font-size: 75%;"
{|  style="font-size: 75%;"
Line 48: Line 57:
|}
|}
-
==Results==
+
Characterization results for the most interesting mutants can be found on [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/Conclusion this page]. For the detailed ''in vitro'' results, please refer to [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data this] page, whereas the ''in vivo'' characterizations are [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Reporter_Systems/tetR#TetR_mutant_characterization there].
 +
 
 +
 
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 19:30, 26 October 2011