Difference between revisions of "Part:BBa K2680063"
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This circuit constitutes a complete IFFL, since heat activates both mScarlet-I (a reporter) and the protease mf-Lon (its inhibitor). | 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 TlpA<sub>39</sub>. Unlike the original TlpA, TlpA<sub>39</sub> undergoes a conformational change at 39C and loses its ability to bind the downstream pTlpA. Therefore, TlpA<sub>39</sub> 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#3b, a medium-strong degradation tag from part series K2333401-K2333406. | + | J23107, a medium-weak constitutive promoter, constitutively expresses TlpA<sub>39</sub>. Unlike the original TlpA, TlpA<sub>39</sub> undergoes a conformational change at 39C and loses its ability to bind the downstream pTlpA. Therefore, TlpA<sub>39</sub> 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#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 <partinfo>BBa_K2680251</partinfo>. |
The production of mf-Lon in the circuit follows a similar process. Increased temperature results in a conformational shape in TlpA<sub>39</sub>, which loses its ability to bind a downstream promoter. However, in the case of mf-Lon, TlpA<sub>39</sub> fails to bind pTlpAr, the reverse complement of pTlpA (pTlpAr is 200-fold weaker than pTlpA). Without repression from TlpA<sub>39</sub>, pTlpAr expresses the mf-Lon protease. An ssra degradation tag follows the mf-Lon in this circuit, resulting in increased turnover of mf-Lon. | The production of mf-Lon in the circuit follows a similar process. Increased temperature results in a conformational shape in TlpA<sub>39</sub>, which loses its ability to bind a downstream promoter. However, in the case of mf-Lon, TlpA<sub>39</sub> fails to bind pTlpAr, the reverse complement of pTlpA (pTlpAr is 200-fold weaker than pTlpA). Without repression from TlpA<sub>39</sub>, pTlpAr expresses the mf-Lon protease. An ssra degradation tag follows the mf-Lon in this circuit, resulting in increased turnover of mf-Lon. | ||
Revision as of 02:54, 18 October 2018
39C Temperature-Sensitive mScarlet-I pdt#3b, with mf-lon ssra
39C Temperature-Sensitive mScarlet-I pdt#3b, with mf-lon ssra. Components: J23107 TPL + ptlpA B0034m mScarlet-I pdt#3b 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#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.
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#3b) and its inhibitor (mf-Lon).
Characterization
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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 4275
Illegal AgeI site found at 4359
Illegal AgeI site found at 4565
Illegal AgeI site found at 4590 - 1000COMPATIBLE WITH RFC[1000]