Revision as of 19:24, 26 October 2011

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Mutant TetRs

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

Strategy

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:

Site-specific mutagenesis , 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)

PCR-induced mutagenesis , which specifically amplifies two halves of the gene of interest from a linear template and introduces a mutation in one of them, followed by a stich-PCR to join the two halves and add the necessary extensions.

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.

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

A summary of the in vitro and in vivo results can be found on this page.

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 {{: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)
@@ 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.
+=== 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%;"
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 |-
 |}
+A summary of the ''in vitro'' and ''in vivo'' results can be found on this page.
 {{:Team:EPF-Lausanne/Templates/Footer}}

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

From 2011.igem.org

Revision as of 19:24, 26 October 2011

Mutant TetRs

Strategy

Mutants obtained