Part:BBa_K2680061

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

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
  
• 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 3305
  Illegal AgeI site found at 3389
  Illegal AgeI site found at 3595
  Illegal AgeI site found at 3620
  
• 1000
  COMPATIBLE WITH RFC[1000]