Composite

Part:BBa_K2066039

Designed by: Kalen Clifton, Christine Gao, Andrew Halleran, Ethan Jones, Likhitha Kolla, Joseph Maniaci, John Marken, John Mitchell, Callan Monette, Adam Reiss   Group: iGEM16_William_and_Mary   (2016-10-14)


Sigma54 Enhancer 57S with 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 loops over to the promoter 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.

This insert has the 57s two tetO binding sites, which allows for three distinct states of output (repressed, intermediate, unrepressed). The genetic circuit inspired by Amit et. al. was PCRed from the enhancer to the end of the mCherry sequence and put onto a UNS Biobrick backbone. This part also includes the double terminator at the end of the mCherry, since there was no evidence of a double terminator labelled in the sequences of the orignal 57s files.

The enhancer, 57s tet cassette, promoter, RBS, NRI, and mCherry sequences are 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 sequences are 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]


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