Difference between revisions of "Part:BBa K2520009"

 
 
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<partinfo>BBa_K2520009 short</partinfo>
 
<partinfo>BBa_K2520009 short</partinfo>
  
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CMV-tTA-hGH
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Tetracycline-controlled transactivator protein (tTA) is composed of the Tet repressor DNA binding protein (TetR) from Escherichia coli fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE).
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In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the target gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.
  
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<!-- Add more about the biology of this part here
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We created a ready-to-use plasmid for tTA expression in mammalian cells under CMV promoter, by adding hGH poly A- a terminator for mammalian cells. This part is an improvement of <partinfo>K1061015</partinfo>.
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===Usage and Biology===
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===Tet-off===
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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.
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[[File:tet off GFP.png|500px|thumb|center|Figure 1: tet off system]]
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2520009 SequenceAndFeatures</partinfo>
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===Tet-on===
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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.
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===Characterization===
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A demonstration of Tet-off system is shown below (figure 1). We co-transfected HEK-293 cells with two plasmids: <partinfo>K2520006</partinfo> - TRE-GFP-hGH and <partinfo>K2520009</partinfo> - 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.
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[[File:induction_curve_tre_tta.png|500px|thumb|center|Figure 1: Weighted median of cells expressing GFP depending on different concentrations of Doxycyline]]
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[[File:tre_histograms.png|500px|thumb|center|Figure 2: Fluorescence intensity of cells expressing GFP in different concentrations of Doxycyline]]
  
  
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===Functional Parameters===
 
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<partinfo>BBa_K2520009 parameters</partinfo>
 
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2520009 SequenceAndFeatures</partinfo>

Latest revision as of 10:00, 28 October 2017


CMV-tTA-hGH

Tetracycline-controlled transactivator protein (tTA) is composed of the Tet repressor DNA binding protein (TetR) from Escherichia coli fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE). In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the target gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.

We created a ready-to-use plasmid for tTA expression in mammalian cells under CMV promoter, by adding hGH poly A- a terminator for mammalian cells. This part is an improvement of BBa_K1061015.

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.

Figure 1: tet off system

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.

Characterization

A demonstration of Tet-off system is shown below (figure 1). We co-transfected HEK-293 cells with two plasmids: BBa_K2520006 - TRE-GFP-hGH and BBa_K2520009 - 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.


Figure 1: Weighted median of cells expressing GFP depending on different concentrations of Doxycyline
Figure 2: Fluorescence intensity of cells expressing GFP in different concentrations of Doxycyline


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1245
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1766