Difference between revisions of "Part:BBa K613013"
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===In vivo characterization=== | ===In vivo characterization=== | ||
| − | This TetR mutant was characterized ''in vivo'' by putting it into [https://parts.igem.org/Part:pSB3K1 pSB3K1] under a constitutive promoter ([https://parts.igem.org/Part:J23116 J23116]). This plasmid was cotransformed with [https://parts.igem.org/Part:J61002 J61002] harbouring RFP under pTet promoter ([https://parts.igem.org/Part:B0040 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. | + | This TetR mutant was characterized ''in vivo'' by putting it into [https://parts.igem.org/Part:pSB3K1 pSB3K1] under a constitutive promoter ([https://parts.igem.org/Part:J23116 J23116]). This plasmid was cotransformed with [https://parts.igem.org/Part:J61002 J61002] harbouring RFP under consensus pTet promoter ([https://parts.igem.org/Part:B0040 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''' | '''Induction curves''' | ||
| + | Fluorescence measurements (RFUs) were normalized by OD600 values. | ||
| − | + | [[Image:EPFL_TetR-V36F-induction.png|600px]] | |
| − | + | ||
| − | [[Image:EPFL_TetR-V36F- | + | With no ATC, the V36F mutant exhibits about 2500 normalized RFUs, which is the same level of expression as 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 than with the wild-type - which expresses 25000 RFUs at the same ATC concentration. The V36F mutant may have an altered ATC binding property. | ||
| Line 76: | Line 78: | ||
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-V36F- | + | [[Image:EPFL_TetR-V36F-doseresponse.png|600px]] |
| + | |||
| + | 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 by ATC than the wild-type protein, where maximal repression correlates with 25000 RFUs. | ||
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Revision as of 18:56, 21 September 2011
TetR V36F mutant
TetR with a point mutation V36F.
In vitro characterization
Using the MITOMI technique we determined the DNA binding landscape of the TetR V36F mutant.
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 |
ΔΔG° values are indicated in kcal/mol
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.
With no ATC, the V36F mutant exhibits about 2500 normalized RFUs, which is the same level of expression as 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 than with the wild-type - which expresses 25000 RFUs at the same ATC concentration. The V36F mutant may have an altered ATC binding property.
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 by ATC than the wild-type protein, where maximal repression correlates with 25000 RFUs.
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]