Part:BBa_K2680052
37C Temperature-sensitive mScarlet pdt#3b
37C temperature-sensitive mScarlet pdt#3b. Components: J23107 ts-CI + R0051 mScarlet-I pdt#3b.
Mechanics
In this circuit, temperature-sensitive CI is expressed constitutively by J23107, a medium-weak 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#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.
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.
Characterization
As shown in the figure above, this circuit is temperature-sensitive and inducible at 37C. The cI repressor cannot bind the cI promoter at this temperature, allowing the expression of mScarlet-I.
When taken out of 37C (gray region) and brought back to 29C, fluorescence decreases as induction of the circuit stops. The cI repressor binds the downstream promoter, repressing the expression of mScarlet-I.
William and Mary iGem attempted to incorporate this circuit into an IFFL by integrating this with circuit BBa_K2680050, a temperature-sensitive mf-Lon circuit also induced at 37C. This forms an IFFL, since 37C activates a reporter (mScarlet-I) and its inhibitor (mf-Lon). However, the IFFL did not satisfy Team William and Mary's goals for their 2018 project, since the IFFL did not create a pulse of fluorescence (see figure below).
The toxicity of mf-Lon in the combined circuits resulted in cell death, as shown below.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 51
Illegal NheI site found at 74
Illegal NotI site found at 1518 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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