Composite

Part:BBa_K2680069

Designed by: Stephanie Do, Xiangyi Fang, Ethan Jones, Jessica Laury, Adam Oliver, Lillian Parr, Chengwu Shen, Tinh Son, Julia Urban, Yashna Verma, Hanmi Zou   Group: iGEM18_William_and_Mary   (2018-10-06)


39C Temperature-Sensitive mScarlet, with mf-Lon ssra

components, J23107 TPL + ptlpA B0034m mScarlet-I thrL terminator + ptlpAr BCD8 mf-Lon ssrA

Mechanics

J23107, a medium-weak constitutive promoter, constitutively expresses TlpA39. Unlike the original TlpA, TlpA39 undergoes a conformational change at 39C and loses its ability to bind the downstream pTlpA. Therefore, TlpA39 halts its repression of pTlpA at temperatures above 39C, inducing the expression of the rapidly-maturing mScarlet-I.

The production of mf-Lon in the circuit follows a similar process. Increased temperature results in a conformational shape in TlpA39, which loses its ability to bind a downstream promoter. However, in the case of mf-Lon, TlpA39 fails to bind pTlpAr, the reverse complement of pTlpA (pTlpAr is 200-fold weaker than pTlpA). Without repression from TlpA39, pTlpAr expresses the mf-Lon protease. An ssra degradation tag follows the mf-Lon in this circuit, resulting in increased turnover of mf-Lon.

As an IFFL

In a complete IFFL (incoherent feed forward loop), an input must activate both a reporter and its inhibitor. Therefore, this circuit requires a pdt degredation tag on the mScarlet-I, allowing for degredation by the mf-Lon protease to function as an IFFL.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 51
    Illegal NheI site found at 74
    Illegal NotI site found at 613
    Illegal NotI site found at 1920
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 898
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1201
    Illegal AgeI site found at 4194
    Illegal AgeI site found at 4278
    Illegal AgeI site found at 4484
    Illegal AgeI site found at 4509
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None