Composite

Part:BBa_K2066116

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 03:02, 29 October 2016 by Cemonette (Talk | contribs)


Synthetic Enhancer Project: 3x TetO Binding Cassette (52s) + sfGFP on UNS

This part takes the synthetic enhancer circuit with a three TetO binding cassette and puts it onto a single BioBrick backbone flanked by the UNS regions. It includes a sfGFP reporter coding sequence downstream of the sigma 54 promoter. This part allows for a four step output response due to the three TetO binding cassette in the spacer region between the enhancer and promoter.The number of filled TetO sites influences the rigidity and the thermodynamics of the looping.

This part should be transformed with the helper plasmid Bba_K2066037 to get a consititutive expression of TetR repressor and NRII2302 (which is a kinase and phosphorylates NRI to activate it and allow it to bind to the enhancer).

Source: The enhancer, tet cassette, glnAp2 synthetic promoter, and NRI 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 sfGFP flourescent reporter design is inspired by C. Lou, B. Stanton, Y.-J. Chen, B. Munsky, C. A. Voigt, Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137 (2012). doi:10.1038/nbt.2401 pmid:23034349. 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
    Illegal NheI site found at 206
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 171
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 951
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1939


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