Part:BBa_K2680069
39C Temperature-Sensitive mScarlet, with mf-Lon ssra
components, J23107 TPL + ptlpA B0034m mScarlet-I thrL terminator + ptlpAr BCD8 mf-Lon ssrA
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 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
- 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 613
Illegal NotI site found at 1920 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 898
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1201
Illegal AgeI site found at 4194
Illegal AgeI site found at 4278
Illegal AgeI site found at 4484
Illegal AgeI site found at 4509 - 1000COMPATIBLE WITH RFC[1000]
| None |