Part:BBa_K2680063

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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As can be seen, the amount of mscarlet-I being degraded by mf-lon is roughly equal to the rate of mscarlet-I being produced. The result is a stagnant level of fluorescence. Because of this, this circuit did not generate the expected IFFL pulse. This outcome falls in line with other circuits that have both mscarlet-I and mf-lon.

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
  Illegal AgeI site found at 4275
  Illegal AgeI site found at 4359
  Illegal AgeI site found at 4565
  Illegal AgeI site found at 4590
  
• 1000
  COMPATIBLE WITH RFC[1000]