Composite

Part:BBa_K2066113

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)
Revision as of 03:05, 29 October 2016 by Lkolla (Talk | contribs)


Synthetic Enhancer with 2X TetO cassette (55as) on UNS backbone

The synthetic enhancer, first characterized by Amit et. al. 2011, allows for a multistate transfer function by modulating the rigidity of the spacer region between the enhancer promoter region, thus affecting the DNA rigidity and hence the kinetic capability of the two components to loop together and initiate transcription.

This synthetic enhancer part consists of a 2x TetO binding cassette within the spacer region between the enhancer and promoter. The two binding sites allows for three discrete states of output: a repressed, intermediate, and unrepressed state. Depending on the steady state of TetR, the amount of bound TetR varies and thus affects the rigidity of the spacer region as well as the kinetics of the looping between the enhancer and promoter, which allows for the initiation of transcription. This part has to be transformed in conjugation with the pACT Tet plasmid (Bba_K2066037), which controls the expression of TetR and NRII2302, a kinase that activates the NRI product (which binds and primes the upstream enhancer region).

Source: The enhancer, tet cassette (55as), glnAp2 synthetic promoter, NRI coding region, and mCherry coding region sequences were derived 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 at the ends of the insert are derived from Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, gkt860. A huge thanks to all the researchers involved in its original creation!

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 111
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 890
  • 1000
    COMPATIBLE WITH RFC[1000]


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