Difference between revisions of "Part:BBa K613019"
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Fluorescence measurements (RFUs) were normalized by OD600 values. | Fluorescence measurements (RFUs) were normalized by OD600 values. | ||
| − | [[ | + | [[Image:EPFL_TetR-P39QY42M-induction.png|600px]] |
In the absence of ATC, RFP expression in presence of the mutant goes up to 25000 normalized RFUs, which is the far more than expression level of the wild-type TetR in the same conditions. This shows that the mutant can only poorly bind and inactivate pTet - in comparison to the wild-type and other mutants such as V36F([https://parts.igem.org/Part:BBa_K613013 K613013]) or V36F W43S([https://parts.igem.org/Part:BBa_K613014 K613014]). With 2000 ng/mL of ATC in the cell culture, RFP expression is further increased, but this change is quite small. This indicates again that the P39Q y42M mutant is not repressing pTet. | In the absence of ATC, RFP expression in presence of the mutant goes up to 25000 normalized RFUs, which is the far more than expression level of the wild-type TetR in the same conditions. This shows that the mutant can only poorly bind and inactivate pTet - in comparison to the wild-type and other mutants such as V36F([https://parts.igem.org/Part:BBa_K613013 K613013]) or V36F W43S([https://parts.igem.org/Part:BBa_K613014 K613014]). With 2000 ng/mL of ATC in the cell culture, RFP expression is further increased, but this change is quite small. This indicates again that the P39Q y42M mutant is not repressing pTet. | ||
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Fluorescence measurements (RFUs) were normalized by OD600 values. For each ATC concentration, we estimated the steady-state fluorescence expression by averaging the measurements over the last hour. | Fluorescence measurements (RFUs) were normalized by OD600 values. For each ATC concentration, we estimated the steady-state fluorescence expression by averaging the measurements over the last hour. | ||
| − | [[ | + | [[Image:EPFL_TetR-P39QY42M-doseresponse.png|600px]] |
The dose-response curve shows that ATC has almost no action on RFP expression. While we cannot know if this mutant still binds ATC in a normal way, it is clear that P39K has only little repressive action on the pTet promoter. | The dose-response curve shows that ATC has almost no action on RFP expression. While we cannot know if this mutant still binds ATC in a normal way, it is clear that P39K has only little repressive action on the pTet promoter. | ||
Revision as of 22:15, 21 September 2011
TetR P39Q Y42M mutant
This is a TetR mutant carrying the P39QY42M double mutation.
In vivo characterization
This TetR mutant was characterized in vivo by putting it into pSB3K1 under a constitutive promoter (J23116). This plasmid was cotransformed with J61002 harbouring RFP under pTet promoter (B0040) in DH5alpha cells. Cells were grown in a medium containing Kanamycin & Amplicillin plus different concentrations of ATC, ranging from 0 to 2000 ng/mL. OD600 absorbence and RFP fluorescence were measured every 10 minutes during 12 hours on a platereader machine.
Induction curves
Fluorescence measurements (RFUs) were normalized by OD600 values.
In the absence of ATC, RFP expression in presence of the mutant goes up to 25000 normalized RFUs, which is the far more than expression level of the wild-type TetR in the same conditions. This shows that the mutant can only poorly bind and inactivate pTet - in comparison to the wild-type and other mutants such as V36F(K613013) or V36F W43S(K613014). With 2000 ng/mL of ATC in the cell culture, RFP expression is further increased, but this change is quite small. This indicates again that the P39Q y42M mutant is not repressing pTet.
Dose-response curve
Fluorescence measurements (RFUs) were normalized by OD600 values. For each ATC concentration, we estimated the steady-state fluorescence expression by averaging the measurements over the last hour.
The dose-response curve shows that ATC has almost no action on RFP expression. While we cannot know if this mutant still binds ATC in a normal way, it is clear that P39K has only little repressive action on the pTet promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]