Composite

Part:BBa_K2680059

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 mf-Lon ssra

39C temperature-sensitive mf-lon ssRA. Components: J23107 TPL + ptlpA BCD8 mf-lon ssrA thrL

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 mf-Lon protease. An ssra tag follows the mf-Lon to increase mf-Lon turnover.

As an IFFL

In an IFFL (incoherent feed-forward loop), an input activates a reporter as well as its inhibitor. This circuit contains the protease mf-Lon, which can degrade and therefore inhibit reporters. To form a complete IFFL, this circuit must be combined with a circuit with a reporter, such as mScarlet-I with a degradation tag.

Characterization

In this figure, heat induces both an mScarlet-I-containing circuit ( a reporter) and BBa_K2680059 (its inhibitor). The combined circuits thus act as an IFFL. However, the fluorescence in this figure continues to increase rather than making the pulse desired by Team William and Mary. William and Mary attributes this continued increase of fluorescence to the weakness of mf-Lon in this particular circuit.

In the figure above, heat again induces an mScarlet-I-containing circuit ( a reporter) and BBa_K2680059 (its inhibitor). Unlike the previous IFFL, this IFFL does not result in increased fluorescence over time. However, it still fails to create the desired fluorescence pulse. Team William and Mary again attributes this to the weakness of mf-Lon. While increasing the strength of mf-Lon was considered and attempted for other circuits, this increase resulted in toxicity and eventual cell death.

Team William and Mary created a successful pulse by chemically inducing the IFFL composed of BBa_K2333428 (an mScarlet-I containing reporter) and BBa_K2680059 (its inhibitor).

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
  • 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 3305
    Illegal AgeI site found at 3389
    Illegal AgeI site found at 3595
    Illegal AgeI site found at 3620
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None