Difference between revisions of "Part:BBa K2066038"

Latest revision as of 21:29, 19 October 2016

Sigma54 Enhancer 57S, no DT, UNS standard

The physical interaction between the assembled sigma 54 promoter complex (glnAp2) and enhancer sites causes the activation of transcription.

Usage and Biology

Amit et. al. generated a genetic circuit regulated by the availability of NRI binding protein as well as the NRII2302 helper protein, which can phosphorylate the NRI protein and activate it as to allow it to bind to the enhancer region. Once this happens, the DNA from the enhancer to the promoter loops and allows for transcription of more NRI (positive feedback) as well as the fluorescent reporter. When repressor binding sites are placed in the spacer region between the enhancer and promoter, this allows for the regulation of output. The TetR binding makes the DNA looping harder and more rigid, resulting in less transcription of the fluorescent reporter. Multiple repressor binding sites can give rise to discrete number of TetR binding to the region at a given time, thus modulating the ability for the DNA to loop, ultimately giving rise to different states of expression depending on the availability of functional apo-repressor protein.

In this insert, there are two tet binding sites in 57s, allowing for three discrete states of output.

Sequences from Amit, R., Garcia, H. G., Phillips, R. & Fraser, S. E. Building enhancers from the ground up: a synthetic biology approach. Cell146, 105–118 (2011). The UNS sequence from Torella et. al.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 111
Illegal NheI site found at 206
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 151
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 951
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 ===Usage and Biology===
-Amit et. al. generated a genetic circuit regulated by the availability of NRI binding protein as well as the NRII2302 helper protein, which can phosphorylate the NRI protein and activate it as to allow it to bind to the enhancer region.  Once this happens, the looping (shown in Figure 1) the DNA from the enhancer to the promoter allows for transcription of more NRI (positive feedback) as well as the fluorescent reporter.  When repressor binding sites are placed in the spacer region between the enhancer and promoter, this allows for the regulation of output.  The TetR binding makes the DNA looping harder and more rigid, resulting in less transcription of the fluorescent reporter.  Multiple repressor binding sites can give rise to discrete number of TetR binding to the region at a given time, thus modulating the ability for the DNA to loop, ultimately giving rise to different states of expression depending on the availability of functional apo-repressor protein.
+Amit et. al. generated a genetic circuit regulated by the availability of NRI binding protein as well as the NRII2302 helper protein, which can phosphorylate the NRI protein and activate it as to allow it to bind to the enhancer region.  Once this happens, the DNA from the enhancer to the promoter loops and allows for transcription of more NRI (positive feedback) as well as the fluorescent reporter.  When repressor binding sites are placed in the spacer region between the enhancer and promoter, this allows for the regulation of output.  The TetR binding makes the DNA looping harder and more rigid, resulting in less transcription of the fluorescent reporter.  Multiple repressor binding sites can give rise to discrete number of TetR binding to the region at a given time, thus modulating the ability for the DNA to loop, ultimately giving rise to different states of expression depending on the availability of functional apo-repressor protein.
 In this insert, there are two tet binding sites in 57s, allowing for three discrete states of output.