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Team:EPF-Lausanne/Our Project/TetR mutants/muTetRs
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* <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) | ||
| - | * <html> <a href="https://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/TetR_Extension_PCR"> PCR-induced mutagenesis </a></html>, 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 | + | 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== | ==What== | ||
Revision as of 01:21, 22 September 2011
Mutant TetRs
In vitro Main | Why TetR? | Mutant TetRs | MITOMI Data | In-vivo & In-vitro outlineHow
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 |
Results
