Coding

Part:BBa_K613014

Designed by: Lilia Salimova   Group: iGEM11_EPF-Lausanne   (2011-09-20)

TetR V36FW43S mutant

This is a TetR mutant, who carries the VF36WS43 mutation. Part of the EPFL2011 muTetR collection.


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.

EPFL TetR-V36FW43S-induction.png

In the absence of ATC, RFP expression in presence of the mutant goes up to 2500 normalized RFUs, which is the same level of expression as for the wild-type TetR. This shows that the mutant is able to bind and inactivate pTet with the same strength as the wild-type. With 2000 ng/mL of ATC in the cell culture, RFP expression rises up to 16000 normalized RFUs. Even if ATC does inhibit TetR function, this inhibition is less striking compared to the wild-type at the same ATC concentration. The V36F W43S mutant may have an altered ATC binding property. Interestingly, the V36FW43S data resemble closely to the V36F mutant K613013; they both have a mutation on the 36 residue.


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.

EPFL TetR-V36FW43S-doseresponse.png

ATC action on the TetR mutant is clearly visible on this graph. From 200 ng/mL, RFP expression is reaching a plateau, indicating that the maximal TetR inhibition has been attained. The mutant is less repressed in presence of ATC than the wild-type protein is.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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