Part:BBa_K2680057
Tight Repression 37C Temperature-Sensitive mf-lon ssra
Tight Repression 37C Temperature-Sensitive mf-lon ssra. Components: J23100 ts-CI+ R0051 mf-Lon ssrA.
Mechanics
Strong promoter J23100 constitutively expresses the CI repressor protein, which binds the downstream CI promoter. However, this circuit contains temperature sensitive CI, which denatures at 37C and consequently loses its ability to bind the CI promoter. Therefore, increasing the temperature to 37C alleviates the repression of the CI promoter and allows for the expression of mf-Lon protease. An ssra degradation tag follows the mf-Lon in this circuit, increasing mf-Lon turnover.
Role in IFFL
In an IFFL (incoherent feed-forward loop), an input activates a reporter and its inhibitor. This circuit produces the mf-Lon protease, which can degrade and thus inhibit promoters like mScarlet-I. Thus, for use in an IFFL, this circuit must be combined with a reporter-containing circuit. A degradation tag, such as a pdt tag, should follow the reporter coding sequence.
Characterization
As shown in the figure above, an IFFL composed of BBa_K2680057 and BBa_K333424 (pTet mscarlet-I-pdt#3) containing circuit created a pulse of fluorescence when chemically induced.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 51
Illegal NheI site found at 74 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2903
Illegal AgeI site found at 2987
Illegal AgeI site found at 3193
Illegal AgeI site found at 3218 - 1000COMPATIBLE WITH RFC[1000]
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