Difference between revisions of "Part:BBa K2680069"

Revision as of 02:14, 18 October 2018

39C Temperature-Sensitive mScarlet, with mf-Lon ssra

components, J23107 TPL + ptlpA B0034m mScarlet-I 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 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

In a complete IFFL (incoherent feed forward loop), an input must activate both a reporter and its inhibitor. Therefore, this circuit requires a pdt degredation tag on the mScarlet-I, allowing for degredation by the mf-Lon protease to function as an IFFL.

Sequence and Features