Difference between revisions of "Part:BBa K2520006"
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| − | [[File:induction_curve_tre_tta.png| | + | [[File:induction_curve_tre_tta.png|400px|thumb|center|Figure 1: Weighted median of cells expressing GFP depending on different concentrations of Doxycyline]] |
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| + | [[File:tre_histograms.png|400px|thumb|center|Figure 2: Fluorescence intensity of cells expressing GFP in different concentrations of Doxycyline]] | ||
Revision as of 13:23, 27 October 2017
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This part is an improvement of Status: 500
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Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84. Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84. Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 85. Compilation failed in require at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12. Compilation failed in require at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9. Compilation failed in require at /websites/parts.igem.org/cgi/lib/Change.pm line 8. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Change.pm line 8. Compilation failed in require at /websites/parts.igem.org/cgi/lib/Part.pm line 12. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Part.pm line 12. Compilation failed in require at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8.
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. This device allows inducible expression of GFP within mammalian cells. The TRE promoter is made up of Tet operator (TetO) sequence concatemers fused to a minimal hCMV. This promoter can be used for inducible expression of the downstream gene in two different schemes.
Tet-off
In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the downstream gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains minimal.
Tet-on
In the presence of Dox, rtTA binds the TRE promoter and activates transcription of the downstream gene. In the absence of Dox, rtTA cannot bind the TRE promoter, and expression of the downstream gene is minimal.
Usage and Biology
In this device, the inducible protein is GFP, and can be used easily for calibration and characterization of inducible systems. The human growth hormone (hGH) terminator has been added to allow for efficient expression of the GFP.
Characterization
A demonstration of Tet-off system is shown below (figure 1). We co-transfected HEK-293 cells with two plasmids: Status: 500 Content-type: text/html
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- TRE-GFP-hGH and Status: 500
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- CMV-tTA-hGH. The transfected cells expressed different levels of GFP depending on Doxycycline concentration, due to the binding of tTA to the TRE promoter. We set a negative controls: co-transfection of TRE-GFP-hGH and pUC19 - for measurement of the basal level of GFP expression without the binding of tTA to the TRE promoter.
Sequence and Features
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Status: 500 Content-type: text/html
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Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84. Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84. Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 85. Compilation failed in require at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12. Compilation failed in require at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9. Compilation failed in require at /websites/parts.igem.org/cgi/lib/Change.pm line 8. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Change.pm line 8. Compilation failed in require at /websites/parts.igem.org/cgi/lib/Part.pm line 12. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Part.pm line 12. Compilation failed in require at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8. BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8.
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