"
Team:EPF-Lausanne/Our Project/TetR mutants/muTetRs
From 2011.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Mutant TetRs}} | {{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Mutant TetRs}} | ||
| - | |||
| Line 10: | Line 9: | ||
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. | 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. | ||
| - | |||
{| style="font-size: 75%;" | {| style="font-size: 75%;" | ||
Revision as of 01:21, 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 , 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.
| 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
