Difference between revisions of "Part:BBa K613018"
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<partinfo>BBa_K613018 short</partinfo> | <partinfo>BBa_K613018 short</partinfo> | ||
| − | This is a TetR mutant | + | This is a TetR mutant that carries the Y42F K108E double mutation. |
| − | + | Part of the <html> <a href="http://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/muTetRs">EPFL2011 muTetR collection. </a> </html> | |
===In vivo characterization=== | ===In vivo characterization=== | ||
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Fluorescence measurements (RFUs) were normalized by OD600 values. | Fluorescence measurements (RFUs) were normalized by OD600 values. | ||
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| + | [[Image:EPFL_TetR-Y42FK108E-induction.png|600px]] | ||
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| + | In the absence of ATC, RFP expression in presence of the mutant goes up to 5000 normalized RFUs, which is slightly more than what is observed with the wild-type or the V36F mutant([https://parts.igem.org/Part:BBa_K613013 K613013]) for example. This indicates that the mutant is able to strongly bind and inactivate pTet, although less than the wild-type. With 2000 ng/mL of ATC in the cell culture, RFP expression rises up to 25000 normalized RFUs - a value observed for the wild-type and the P39K mutant([https://parts.igem.org/Part:BBa_K613016 K613016]) - showing that the P39K mutant is repressed by ATC in a normal way. | ||
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'''Dose-response curve''' | '''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. | 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. | ||
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| + | [[Image:EPFL_TetR-Y42FK108E-doseresponse.png|600px]] | ||
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| + | The graph shows that the Y42F K108E double mutant is well repressed by ATC. RFP expression is reaching a plateau from 200 ng/mL ATC, indicating that TetR repression is maximal. | ||
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Latest revision as of 22:07, 21 September 2011
TetR Y42F K108E mutant
This is a TetR mutant that carries the Y42F K108E double 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.
In the absence of ATC, RFP expression in presence of the mutant goes up to 5000 normalized RFUs, which is slightly more than what is observed with the wild-type or the V36F mutant(K613013) for example. This indicates that the mutant is able to strongly bind and inactivate pTet, although less than the wild-type. With 2000 ng/mL of ATC in the cell culture, RFP expression rises up to 25000 normalized RFUs - a value observed for the wild-type and the P39K mutant(K613016) - showing that the P39K mutant is repressed by ATC in a normal way.
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 graph shows that the Y42F K108E double mutant is well repressed by ATC. RFP expression is reaching a plateau from 200 ng/mL ATC, indicating that TetR repression is maximal.
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]