Composite

Part:BBa_K2066122

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 22:43, 19 October 2016 by Lkolla (Talk | contribs)


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

This part takes the synthetic genetic circuit from Amit et. al. 2011, the NRII promoter and coding region from Amit et. al. 2011 as well as the TetR promoter and coding region from Bba_I739001 and puts it onto a BioBrick backone flanked by the UNS regions. Used for the synthetic enhancer project, this part consists of an enhancer region, that when bound to phosphorylated NRI protein, can bind to the promoter after the DNA loops to allow for these kinetics. The promoter and coding sequence for the NRII protein (which is mutated to be solely a kinase that can phosphorylate and activate the NRI and allow it to bind to the enhancer region) was taken from the Amit et. al. pACT Tet helper plasmid and put the sequence between the UNS 3 sequence. Furthermore, the Bba_I739001 sequence was inserted into the UNS2 region of the insert to let TetR consititutively expressed by the J23105 promoter. NRII and TetR were moved to the same plasmid as the synthetic enhancer to 1. reduce the circuit's dependence on LacI (which is also expressed in the original pACT Tet helper plasmid) as well as decrease the interference of these plasmids after transformations with the current bacterial circuitry and thus reduce the metabolic strain on the bacteria.

Once the looping and interaction between the enhancer and promoter happens, transcription of the NRI protein for positive feedback as well as the output fluorescent reporter(sfGFP) is initiated. This part (52s) is from the synthetic enhancer genetic circuits created by Amit et. al where there is a tetR binding cassette with three TetO binding sites in the spacer region between the enhancer and the promoter. The three binding sites allows for four discrete states of output: a repressed, two intermediate, and unrepressed states. Small chemical induction with aTc binds to and inactivates available TetR repressor. When a small amount of aTc is present, there isn't enough aTc to find TetR proteins and there is enough repressor available to allow for a constant filling of the three available TetO binding sites. When all TetO sites are bound, the DNA becomes very rigid and makings looping of the enhancer to the promoter extremely difficult, thus not allowing for very minimal transcription of the NRI and reporter proteins. When more aTc is added to the system, more TetR is bound and inactivated and this creates two intermediate steps where enough TetR is bound that at any point only one or two TetR are bound to the teto sites and this decreases the flexibility of the DNA and allows for looping and activation and reporter output less frequently. Finally, when aTc concentration is high, you reach a saturation point where almost all TetR repressors are bound and inactivated and you get maximal amount of looping and thus maximal amount of NRI and reporter transcribed and translated.

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!

Sequence and Features


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


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