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Team:EPF-Lausanne/Our Project/TetR mutants/Conclusion
From 2011.igem.org
In Vivo & In Vitro Outline
In vitro Main | Why TetR? | Mutant TetRs | MITOMI Data | In-vivo & In-vitro outline
For some of the TetR mutants, we managed to have both an in vitro and in vivo characterization. The results are available in the table below.
| Mutant | Biobrick number | Affinity compared to WT - in vivo | Affinity compared to WT - MITOMI | ATC repression |
|---|---|---|---|---|
| V36F | BBa_K613013 | same | higher | altered |
| V36F W43S | BBa_K613014 | same | not tested | altered |
| E37A W43S T141A | BBa_K613015 | same | altered | altered |
| P39K | BBa_K613016 | no affinity | no affinity | normal |
| Y42F K108E | BBa_K613018 | reduced | not tested | normal |
| P39Q Y42M | BBa_K613019 | no affinity | not tested | normal |
The P39K mutant shows in both cases (in vivo and in vitro) no affinity to the Ptet consensus sequence, showing that our results are consistent between the two characterizations.
The V36F mutant either shows a stronger or a similar binding affinity compared to the WT. The differences not being striking, our characterization methods are here again consistent, with the in vitro technique being more sensitive than the in vivo.
Finally, the E37A W43S T141A triple mutant was shown in vitro to have a different consensus sequence that the wild-type Ptet sequence. However, the in vivo experiment only tested the mutant with the wild-type Ptet and shows an affinity in the range of the wild-type TetR. These two results indicate that, althoud a change in specificity, there is still crosstalk with the original Ptet sequence. This mutant is the most promising of the three characterized in our two systems, harbouring a change in specificity as well as DNA binding properties. Still, this is not an orthogonal mutant; more TetRs need to be tested before fully eliminating crosstalk with wild-type Ptet.
The results coming from the in vivo and in vitro part are consistently related. We have tested only 3 mutants in both systems ; we would need more characterization results to improve our selection systems, especially the in vivo part.
The MITOMI results can be found here. The in vivo characterizations are on this page.
