Difference between revisions of "Part:BBa K415506"

Revision as of 03:54, 29 October 2010

Figure 1. Schematic depicting the schematic of the TREt system.

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. (Figure 1.)

Characterization

Figure 2. Comparison of TREt and EGSH reporter constructs.

Comparison of TREt and EGSH Promoter Constructs

In Figure 2, 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.

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 1: / Line 1: @@
 __NOTOC__
+[[Image:TRET-logic.png|thumb|right|Figure 1. Schematic depicting the schematic of the TREt system.]]
 <partinfo>BBa_K415506 short</partinfo>
-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.
+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. (Figure 1.)
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 ==Characterization==
-[[Image:Tret_vs_egsh.jpg|thumb|left|Figure 1. Comparison of TREt and EGSH reporter constructs.]] '''Comparison of TREt and EGSH Promoter Constructs'''
+[[Image:Tret_vs_egsh.jpg|thumb|left|Figure 2. 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:[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.
+In Figure 2, 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.
 '''Results'''