Composite

Part:BBa_K2066122:Design

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 05:20, 29 October 2016 by Lkolla (Talk | contribs) (→‎Design Notes)

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Synthetic Enhancer Project: 3X TetO Binding Cassette(52S) + NRII + TetR + sfGFP on UNS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 27
    Illegal NheI site found at 50
    Illegal NheI site found at 194
    Illegal NheI site found at 289
    Illegal NotI site found at 3862
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 254
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1034
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2022


Design Notes

We designed the part to include the synthetic enhancer suite, the NRII helper protein, and constitutively expressed TetR on the same UNS backbone. Making one plasmid reduces the metabolic strain on the cell and further decouples the synthetic enhancer system from LacI/IPTG to allow for minimal inference of our circuits in the complete biological system. We switched the original synthetic enhancer reporter (mCherry) with sfGFP for stronger fluorescent signals. We chose to include UNS overhangs before and after our insert to allow for an ease of clone. UNS sequences are from Torella et. al. Furthermore, the NRII sequence and the synthetic enhancer suite sequences are from Amit et. al. The sfGFP sequence is from Lou et. al. 2012.

Source

The enhancer, tet cassette, glnAp2 synthetic promoter, and NRI coding region sequences were derived from the synthetic enhancer circuits 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 sequence for the TetR controlled by J23105 promoter is from Biobrick part Bba_I739001. The TetR is inserted between the UNS2 sequence and is added to the same plasmid as the synthetic enhancer suite and NRII2302 to reduce the interference with other genetic circuitry in the bacteria and thus decrease overall metabolic strain. 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!

References