Composite

Part:BBa_K2066120

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 04:39, 29 October 2016 by Lkolla (Talk | contribs)


Synthetic Enhancer Project: 3X TetO Binding Cassette(52S) + NRII + sfGFP on UNS 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. For more information on the mechanism, please refer to: http://2016.igem.org/Team:William_and_Mary/Synthetic_Enhancer.

This part takes the synthetic enhancer circuit with a three TetO binding cassette as well as the promoter and coding region of NRII from the helper plasmid of Amit et. al. 2011 circuits and puts it onto a single BioBrick backbone flanked by the UNS regions. 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. Combining the NRII and synthetic enhancer reduces metabolic strain, decouples the system from LacI/IPTG dependence, and allows for an ease of cloning due to the UNS sequences. The number of filled TetO sites influences the rigidity and the thermodynamics of the looping. Furthermore, we included an sfGFP reporter for more pronounced fluorescent signals.

This part should be transformed with Bba_K2066022 to get constitutive expression of TetR repressor and allow for the circuit to output a 4 step multimodal response.

Source: The enhancer, tet cassette, 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 NRII2302 coding region and the promoter that it is controlled by is derived from the helper plasmid pACT tet from Amit et. al 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
    Illegal NotI site found at 3779
  • 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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