Difference between revisions of "Part:BBa K613014"

(→‎In vivo characterization)
(→‎In vivo characterization)
 
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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.  
 
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.  
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Interestingly, the V36FW43S data resemble closely to the V36F mutant [https://parts.igem.org/Part:BBa_K613013 K613013].
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Interestingly, the V36FW43S data resemble closely to the V36F mutant [https://parts.igem.org/Part:BBa_K613013 K613013]; they both have a mutation on the 36 residue.
  
  

Latest revision as of 22:32, 21 September 2011

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]