Difference between revisions of "Part:BBa K415506"

Revision as of 03:33, 29 October 2010

pTRE-Tight L4R1 MammoBlock

Modified TRE promoter with six tetO sites to provide low basal transcriptional activity, a significant improvement over the previously used simple TRE promoter. Upon induction with rtTA or rtTA variants, and the addition of doxycycline (DOX), the transcriptional activity of TREt drastically increases.

Characterization

Figure 1. Comparison of TREt and EGSH reporter constructs.

Comparison of TREt and EGSH Promoter Constructs

In Figure 1, the inducibility of TREt and another inducible promoter, EGSH (Link:[1]), are compared. HEK293 FT cells were infected with reporter constructs for each system. The EGSH system is inducible with ponasterone, and expresses EGFP when induced. The TREt system controls expression of EYFP. Addition of DOX leads to activation of rtTA3, which then induces TREt_EYFP. Controls without inducing chemical factors are shown for both systems. For the EGSH system, (A) indicates the absence and (B) indicates the presence of ponesterone. For the TREt system, (C) indicate the absence and (D) indicates the presence of DOX. Scale bars (red) are 100 μm.

Results Negative control experienced very low level of basal transcription, or "leaky" expression. DOX addition led to dramatic increase in transcriptional activity. This makes the TREt system ideal for inducible, high level expression of proteins, especially lethal or harmful proteins that must be tightly regulated. The TREt system is also widely used in synthetic systems for its rigorous positive feedback loop. tret_vs_egsh.jpg

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal XbaI site found at 30
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal XbaI site found at 30
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal XbaI site found at 30
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 ==Characterization==
-[[Image:TREt_characterization.jpg|thumb|left|Figure 1. DOX Induction of TREt Promoter Post Calcium Phosphate Transfection of HEK293 rtTA3 Cell Line.]] Response of K415506 to DOX induction.
-DOX addition occured with medium change post calcium phosphate transfection of HEK293 rtTA3 cell line with TREt_EGFP construct.
+[[Image:Tret_vs_egsh.jpg|thumb|left|Figure 1. Comparison of TREt and EGSH reporter constructs.]] '''Comparison of TREt and EGSH Promoter Constructs'''
-'''Results'''
+In Figure 1, the inducibility of TREt and another inducible promoter, EGSH (Link:[https://parts.igem.org/Part:BBa_K415507]), are compared. HEK293 FT cells were infected with reporter constructs for each system. The EGSH system is inducible with ponasterone, and expresses EGFP when induced. The TREt system controls expression of EYFP. Addition of DOX leads to activation of rtTA3, which then induces TREt_EYFP. Controls without inducing chemical factors are shown for both systems. For the EGSH system, (A) indicates the absence and (B) indicates the presence of ponesterone. For the TREt system, (C) indicate the absence and (D) indicates the presence of DOX. Scale bars (red) are 100 μm.
-DOX addition led to dramatic increase in transcriptional activity, as seen in Figure 1. Basal transcription level is shown to be low in the left panel.
+'''Results'''
+Negative control experienced very low level of basal transcription, or "leaky" expression. DOX addition led to dramatic increase in transcriptional activity. This makes the TREt system ideal for inducible, high level expression of proteins, especially lethal or harmful proteins that must be tightly regulated. The TREt system is also widely used in synthetic systems for its rigorous positive feedback loop.
+tret_vs_egsh.jpg
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 ==Sequence and Features==
 <partinfo>BBa_K415506 SequenceAndFeatures</partinfo>