Composite

Part:BBa_K2680053

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)


Tight Repression 37C Temperature-Sensitive mScarlet-I pdt#3

Tight Repression 37C Temperature-Sensitive mScarlet-I pdt#3. Components: J23100 ts-CI + R0051 mScarlet-I pdt#3

Mechanics

In this circuit, temperature-sensitive CI is expressed constitutively by J23100, a strong constitutive promoter. However, the CI repressor protein denatures at 37C, losing its ability to bind the downstream CI promoter. Therefore, temperatures above 37C alleviate the repression of the CI promoter, allowing for free 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#3, the strongest degradation tag from part series BBa_K2333001-Bba_K2333006. To view a figure that displays the degradation rates of mScarlet-I with various pdt tags (relative to untagged mScarlet-I), see BBa_K2680251.

Role in 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.

T--William_and_Mary--024.png

As can be seen in the above figure, when combined with a J23100 ts-cI repressed mf-Lon, the amount of mf-Lon produced, relative to the amount of m-scarlet produced, is too high, so no IFFL like pulse is observed.

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 1518
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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