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Team:EPF-Lausanne/Our Project/TetR mutants/muTetRs
From 2011.igem.org
Mutant TetRs
In vitro Main | Why TetR? | Mutant TetRs | MITOMI Data | In-vivo & In-vitro outlineStrategy
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 |
Characterization results for the most interesting mutants can be found on this page. For the detailed in vitro results, please refer to [ this] page, whereas the in vivo characterizations are [ there].
