Difference between revisions of "Part:BBa K2100004"
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<partinfo>BBa_K2100004 short</partinfo> | <partinfo>BBa_K2100004 short</partinfo> | ||
− | + | This construct is an entry vector containing the TRE promoter. The promoter is structured as six tetO sites upstream of a minimal CMV promoter. The induction of doxycycline activates a tetracycline transactivator protein which in turn binds to the tetO sites to initiate transcription of the gene. | |
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K2100004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2100004 SequenceAndFeatures</partinfo> | ||
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+ | We characterized our TRE promoter induced with a fine sweep of Doxycycline from a concentration of 0 uM to 2000 uM. We transfected HEK293 cells with 250ng of TRE-mKate and 250 ng hEF1a-eYFP. We analyzed fluorescent output in the PE Texas Red channel to measure the activity of the TRE promoter under different DOX concentrations. We used 0 uM of DOX as a control. | ||
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+ | https://static.igem.org/mediawiki/parts/9/96/T--MIT--TRE.png | ||
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+ | The results indicate the optimal DOX concentration was 500 uM for maximum TRE activity. Beyond that point, adding a higher concentration of DOX does not significantly increase fluorescent output from the TRE-mKate construct. | ||
Latest revision as of 23:06, 28 October 2016
pENTR pTRE
This construct is an entry vector containing the TRE promoter. The promoter is structured as six tetO sites upstream of a minimal CMV promoter. The induction of doxycycline activates a tetracycline transactivator protein which in turn binds to the tetO sites to initiate transcription of the gene.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal XbaI site found at 30 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal XbaI site found at 30 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal XbaI site found at 30 - 1000COMPATIBLE WITH RFC[1000]
We characterized our TRE promoter induced with a fine sweep of Doxycycline from a concentration of 0 uM to 2000 uM. We transfected HEK293 cells with 250ng of TRE-mKate and 250 ng hEF1a-eYFP. We analyzed fluorescent output in the PE Texas Red channel to measure the activity of the TRE promoter under different DOX concentrations. We used 0 uM of DOX as a control.
The results indicate the optimal DOX concentration was 500 uM for maximum TRE activity. Beyond that point, adding a higher concentration of DOX does not significantly increase fluorescent output from the TRE-mKate construct.