Difference between revisions of "Part:BBa K2680061"
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<partinfo>BBa_K2680061 short</partinfo> | <partinfo>BBa_K2680061 short</partinfo> | ||
− | 39C Temperature-Sensitive (tlpAr) mf-lon ssra. Components: J23107 TPL + ptlpAr BCD8 mf-lon ssrA thrL | + | 39C Temperature-Sensitive (tlpAr) mf-lon ssra. |
+ | Components: J23107 TPL + ptlpAr BCD8 mf-lon ssrA thrL | ||
+ | |||
+ | ===Mechanics=== | ||
+ | J23107 (a medium-weak constitutive promoter) constitutively expresses TlpA<sub>39</sub>, a repressor protein that undergoes a conformational change at 39C. Due to this change, the repressor protein loses its ability to bind the downstream promoter, pTlpAr. Increasing temperature to 39C thus alleviates the repression on pTlpAr, allowing expression of mf-Lon protease. However, pTlpAr is 200-fold weaker than its reverse complement, pTlpA. As well, an ssra tag follows the mf-Lon increase 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. | ||
+ | |||
+ | ===Characterization=== | ||
+ | |||
+ | https://static.igem.org/mediawiki/2018/e/e9/T--William_and_Mary--033_%2B_038.png | ||
+ | |||
+ | As can be seen in the figure above, this mf-lon combined with ptplA B0034 mScarlet-I pdt#3 do not provide the right set of parameters needed to create an IFFL circuit. As a result, there is no visible pulse in fluorescence. | ||
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Latest revision as of 02:06, 18 October 2018
39C Temperature-Sensitive (tlpAr) mf-lon ssra
39C Temperature-Sensitive (tlpAr) mf-lon ssra. Components: J23107 TPL + ptlpAr BCD8 mf-lon ssrA thrL
Mechanics
J23107 (a medium-weak constitutive promoter) constitutively expresses TlpA39, a repressor protein that undergoes a conformational change at 39C. Due to this change, the repressor protein loses its ability to bind the downstream promoter, pTlpAr. Increasing temperature to 39C thus alleviates the repression on pTlpAr, allowing expression of mf-Lon protease. However, pTlpAr is 200-fold weaker than its reverse complement, pTlpA. As well, an ssra tag follows the mf-Lon increase 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.
Characterization
As can be seen in the figure above, this mf-lon combined with ptplA B0034 mScarlet-I pdt#3 do not provide the right set of parameters needed to create an IFFL circuit. As a result, there is no visible pulse in fluorescence.
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 - 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 3305
Illegal AgeI site found at 3389
Illegal AgeI site found at 3595
Illegal AgeI site found at 3620 - 1000COMPATIBLE WITH RFC[1000]