Difference between revisions of "Part:BBa K415507"

Revision as of 03:40, 29 October 2010

pEGSH L4R1 MammoBlock Entry Vector

Figure 1. Schematic of PonS Induction of the EGSH promoter, leading to transcriptional activation.

Schematic: Response of K415507 to the addition of PonS, in the presence of VgEcR and RxR.

L4R1 MammoBlock entry vector. EGSH is an inducible promoter. In the presence of the proteins retinoid X receptor (RxR) and ecdysone receptors (VgEcR), the addition of ponasterone S (PonS) leads to complex formation of RxR, VgEcR, and PonS. This activated complex is then capable of inducing the EGSH promoter and allows for dramatic upregulation in transcriptional activity.

Hef1a promoter leads to low level, constitutive expression of the two halves (VgEcR and RxR) of a receptor responsive to the chemical ponasterone A (PonS).
VgEcR, RxR, and PonS combine to form an active complex.
EGSH is induced by the VgECR-RxR-PonS complex.

Characterization

Figure 3. Transcriptional Upregulation of EGSH in Response to PonS addition in the presence of EgVcR and RxR.

HEK293FT cell lines actively transcribing VgEcR, RxR, and Hygromycin under the low level constitutive promoter hEF1a were transfected with EGSH_EGFP constructs via calcium phosphate transient transfection. The micrographs shown in Figure 3 show characterization data for the cell line in the absence and presence of PonS.

Results

Introduction of PonS significantly upregulates the transcription activity of the EGSH_EGFP construct. EGSH_EGFP is shown to have very low basal expression rate and high fold change in transcription post PonS induction. This property making it ideal for use in sensitive toggles, expression of lethal or harmful proteins, as well as many other applications in genetic engineering and synthetic biology.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 671
Illegal PstI site found at 485
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 671
Illegal PstI site found at 485
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 671
Illegal BglII site found at 115
Illegal BamHI site found at 663
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 671
Illegal PstI site found at 485
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 6
Illegal EcoRI site found at 671
Illegal PstI site found at 485
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 646

@@ Line 6: / Line 6: @@
 L4R1 MammoBlock entry vector. EGSH is an inducible promoter. In the presence of the proteins retinoid X receptor (RxR) and ecdysone receptors (VgEcR), the addition of ponasterone S (PonS) leads to complex formation of RxR, VgEcR, and PonS. This activated complex is then capable of inducing the EGSH promoter and allows for dramatic upregulation in transcriptional activity.
-&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;&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;
+# Hef1a promoter leads to low level, constitutive expression of the two halves (VgEcR and RxR) of a receptor responsive to the chemical ponasterone A (PonS).
+# VgEcR, RxR, and PonS combine to form an active complex.
+# EGSH is induced by the VgECR-RxR-PonS complex.
+&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;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 ==Characterization==
 [[Image:EGSH_characterization.jpg|thumb|left|Figure 3. Transcriptional Upregulation of EGSH in Response to PonS addition in the presence of EgVcR and RxR.]] HEK293FT cell lines actively transcribing VgEcR, RxR, and Hygromycin under the low level constitutive promoter hEF1a were transfected with EGSH_EGFP constructs via calcium phosphate transient transfection. The micrographs shown in Figure 3 show characterization data for the cell line in the absence and presence of PonS.