Difference between revisions of "Part:BBa K2680064"

Revision as of 01:55, 18 October 2018

39C Temperature-Sensitive mScarlet pdt#3d, with mf-Lon ssra

39C Temperature-Sensitive mScarlet pdt#3d, with mf-Lon ssra. Components: J23107 TPL + ptlpA B0034m mScarlet-I pdt-3d thrL terminator + ptlpAr BCD8 mf-Lon ssrA

Mechanics

This circuit constitutes a complete IFFL, since heat activates both mScarlet-I (a reporter) and the protease mf-Lon (its inhibitor).

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 mScarlet-I in this circuit has a pdt degradation tag, allowing for degradation by the mf-Lon protease. Specifically, this circuit contains pdt#3d, the weakest degradation tag from part series K2333401-K2333406.

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

This circuit constitutes a complete IFFL, a circuit where an input activates both a reporter (the mScarlet-I pdt#3d) and its inhibitor (mf-Lon).

Characterization

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Sequence and Features