Revision as of 01:09, 22 September 2011

Mutant TetRs

In vitro Main | Why TetR? | Mutant TetRs | MITOMI Data | In-vivo & In-vitro outline

We used two distinct strategies to make the mutants:

Site-specific mutagenesis , that consists of plasmid amplification step with both forward and reverse primers containing the targeted mutation and the selection step (enzymatic digestion of the methylated dsDNA)

TetR variants	Mutagenesis	Promoter	Cterminal	DNA form	Expression	MITOMI vs 1-off	Biobrick	Sequenced	Sent to registry
V36F	site directed	SP6	eGFP	plasmid	worked well	X	BBa_K613013
V36FW43S	PCR-induced	T7	His-tag	linear	(ins C-term), ITT tested		BBa_K613014
E37AW43ST141A	PCR-induced	T7	His-tag	linear	ITT tested	X	BBa_K613015	X	X
P39K	PCR-induced	T7	His-tag	template	ITT tested, worked	X	BBa_K613016	X	X
Y42F	PCR-induced	T7	His-tag	linear	ITT tested	X	BBa_K613017	X	X
Y42FK108E	PCR-induced	T7	His-tag	linear	ITT tested		BBa_K613018	X	X
P39QY42M	site directed	SP6	eGFP	plasmid	worked well	X	BBa_K613019
P39QY42ML197S	PCR-induced	T7	His-tag	linear	ITT tested		BBa_K613020
P39QY42ML52P	PCR-induced	T7	His-tag	linear	ITT tested		BBa_K613021
E37AP39K	site directed	T7	His-tag	linear			BBa_K613022	X
E37AP39QY42F	site directed	T7	His-tag	linear			BBa_K613023	X
WT	none	T7	His&GFP	both

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 ==How==
-First we chose the <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/Site-specific_mutagenesis"> Site-specific mutagenesis </a></html> approach to make the mutants of TetR.
+We used two distinct strategies to make the mutants:
-But that strategy <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Notebook/July2011#Tuesday.2C_12_July_2011"> was not working well </a> </html> and another one was tried in parallel: <html> <a href="http:/2011.igem.org/Team:EPF-Lausanne/Protocols/TetR_Extension_PCR" PCR-induced mutagenesis.</a></html>
+* <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/Site-specific_mutagenesis"> Site-specific mutagenesis </a></html>, that consists of plasmid amplification step with both forward and reverse primers containing the targeted mutation and the selection step (enzymatic digestion of the methylated dsDNA)
+* <html> <a href="http:/2011.igem.org/Team:EPF-Lausanne/Protocols/TetR_Extension_PCR" PCR-induced mutagenesis </a></html> tha
 ==What==