Part:BBa_K613013
TetR V36F mutant
TetR with a point mutation V36F. Part of the EPFL2011 muTetR collection.
In vitro characterization
Using the MITOMI technique we determined the DNA binding landscape of the TetR V36F mutant. To do so, first we designed and generated the library of double stranded DNA sequences that cover all possible single base substitution within the tetO operator sequence. Based on that library we measured the dissociation constants of the mutant to variable tetO-like sequences and determined the specificity of the mutant to the tet operator sequence (expressed as a PWM).
WebLogo we obtained for the V36F mutant:
Workman CT, Yin Y, Corcoran DL, Ideker T, Stormo GD, Benos PV. enoLOGOS: a versatile web tool for energy normalized sequence logos. Nucleic Acids Res. 2005 Jul 1;33:W389-92.
Position Weight Matrix
| PO | A | T | C | G |
| 1 | 0.164072 | 0.519334 | 0 | 0.22966 |
| 2 | 0.642569 | 0.164072 | 0.188965 | 0.943314 |
| 3 | 1.04481 | 0.558693 | 0.164072 | 0.848264 |
| 4 | 0.959646 | 0.164072 | 2.17536 | 0.718959 |
| 5 | 0.164072 | 1.19894 | 1.79469 | 1.56685 |
| 6 | 1.46463 | 0.164072 | 1.74384 | 1.54845 |
| 7 | 0.966397 | 1.07557 | 0.164072 | 1.72883 |
| 8 | 0.164072 | 0.504312 | 0.755195 | 0.148902 |
| 9 | 0.0876645 | 0.164072 | 0 | 0.0156484 |
| 10 | 0.289722 | 0.164072 | 0.00707563 | 0.843474 |
| 11 | 1.77568 | 1.3447 | 2.34804 | 0.164072 |
| 12 | 0.164072 | 1.72354 | 1.62782 | 1.49176 |
| 13 | 0.877518 | 0.164072 | 1.30879 | 1.9052 |
| 14 | 0.164072 | 0.544642 | 0.3387 | 1.7537 |
| 15 | 0.540091 | 0.8821 | 1.0861 | 0.164072 |
| 16 | 0 | 0.280358 | 0.442769 | 0.164072 |
| 17 | 0.31238 | 0 | 0.164072 | 0 |
Each row represents the changes in binding energy, ΔΔG, compared to the reference sequence upon the substitution to the indicated nucleotide at certain position within the target DNA element. Values are indicated in kcal/mol.
We compared the measured DNA binding affinities of the V36F mutant to the affinities obtained for the wt-TetR and found that V36F mutant interacts with the tetO as strong as the wild-type variant.
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 consensus 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 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 inhibiton is less striking compared to the wild-type at the same ATC concentration. The V36F mutant may have an altered ATC binding property. Interestingly, the V36F W43S data resemble closely to the V36FW43S mutant K613014.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |