Composite

Part:BBa_K2680066

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 (ptplA) mScarlet-I pdt#3b

Components: J23107 TPL + ptplA B0034 mScarlet-I pdt#3b

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 promoter pTlpA at temperatures above 39C, inducing the expression of the rapidly-maturing mScarlet-I. The mScarlet-I in this circuit has a pdt degradation tag, allowing for degradation by the mf-Lon protease. Specifically, this circuit contains pdt#3b, a medium-strong degradation tag from part series K2333401-K2333406. To view a figure that displays the degradation rates of mScarlet-I with various pdt tags (relative to untagged mScarlet-I), see BBa_K2680251.

As an IFFL

In this circuit, heat activates a reporter, mScarlet-I. In a complete IFFL (incoherent feed forward loop), an input must activate both a reporter and its inhibitor. Therefore, this circuit requires mf-Lon (or a different inhibitor of mScarlet-I) to act as an IFFL.

Characterization

"T--William_and_Mary--BBa_K2680059weakmf.png"

In this figure, heat induces both BBa_K268066 and a circuit containing mf-Lon, 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.

"T--William_and_Mary--BBa_K2680058heatfail.png"


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
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None