Difference between revisions of "Part:BBa K2680062"
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===Mechanics===
 
===Mechanics===
  
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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#3, the strongest degradation tag from part series K2333401-K2333406 or <bbpart>K2333401</bbpart>.
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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#3, the strongest 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>.
  
 
===As an IFFL===
 
===As an IFFL===
  
 
In this circuit, heat activates a reporter, mScarlet-I. In a complete IFFL (incoherent feed forward loop), an input must activate both a reporter and its inhibitor. Therefore, this circuit requires mf-Lon (or a different inhibitor of mScarlet-I) to act as an IFFL.
 
In this circuit, heat activates a reporter, mScarlet-I. In a complete IFFL (incoherent feed forward loop), an input must activate both a reporter and its inhibitor. Therefore, this circuit requires mf-Lon (or a different inhibitor of mScarlet-I) to act as an IFFL.
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===Characterization===
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<html><img src="https://static.igem.org/mediawiki/2018/4/4c/T--William_and_Mary--034%2B030.png" width="550px"/></html>
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When combined with BBa_K2680058 (pTLPA mf-lon), it appears that the expression of mf-lon is way too strong for the amount of mscarlet-I produced by BBa_K2680062, resulting in a decrease in fluorescence levels as compared to initial expression.
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<html><img src="https://static.igem.org/mediawiki/2018/5/58/T--William_and_Mary--034%2B031.png" width="550px"/></html>
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When combined with BBa_K2680059 (pTLPA mf-lon-ssra), the amount of mf-lon produced still appears to be a bit too much for the amount of mscarlet-I produced, resulting in stagnant fluorescence levels.
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<html><img src="https://static.igem.org/mediawiki/2018/5/5f/T--William_and_Mary--034%2B032_fixed.png" width="550px"/></html>
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When combined with BBa_K2680060(pTLPAr mf-lon), the amount of mf-lon expressed now appears to be too low as compared to the amount of mscarelt-I produced by BBa_K2680062, as the levels of fluorescence begin to increase exponentially.
  
 
<!-- Add more about the biology of this part here
 
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Latest revision as of 03:09, 18 October 2018


39C Temperature-Sensitive mScarlet-I pdt#3

39C Temperature-Sensitive mScarlet-I pdt#3. Components: J23107 TPL + ptlpA b0034m mScarlet-I pdt#3

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 mScarlet-I in this circuit has a pdt degradation tag, allowing for degradation by the mf-Lon protease. Specifically, this circuit contains pdt#3, the strongest 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.

As an IFFL

In this circuit, heat activates a reporter, mScarlet-I. In a complete IFFL (incoherent feed forward loop), an input must activate both a reporter and its inhibitor. Therefore, this circuit requires mf-Lon (or a different inhibitor of mScarlet-I) to act as an IFFL.

Characterization

When combined with BBa_K2680058 (pTLPA mf-lon), it appears that the expression of mf-lon is way too strong for the amount of mscarlet-I produced by BBa_K2680062, resulting in a decrease in fluorescence levels as compared to initial expression.

When combined with BBa_K2680059 (pTLPA mf-lon-ssra), the amount of mf-lon produced still appears to be a bit too much for the amount of mscarlet-I produced, resulting in stagnant fluorescence levels.

When combined with BBa_K2680060(pTLPAr mf-lon), the amount of mf-lon expressed now appears to be too low as compared to the amount of mscarelt-I produced by BBa_K2680062, as the levels of fluorescence begin to increase exponentially.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 898
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1201
  • 1000
    COMPATIBLE WITH RFC[1000]