Mutant TetRs

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

How

We used two distinct strategies to make the 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.

What

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

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

From 2011.igem.org

Mutant TetRs

How

What

Results