Part:BBa_K4244023

placUV5:ccdA + pcspA:ccdB

The pcspA (BBa_K4244021) is a constitutive promoter that has a high rate of transcription but contains a long 5′ UTR of 159 bp that, at 37°C, acquires an unstable secondary structure and leads to its degradation. On the other hand, at lower temperatures it forms a stable configuration, which allows translation. In this case, a toxin (ccdB) under the cspA promoter is expressed but counteracted by the antitoxin ccdA (BBa_K4244023), which in turn is expressed by being placed after the constitutive LacUV5 (BBa_M36801) [1]. At lower temperature the cspA stabilizes and ccdB overexpresses ccdA, which leads to cell death.

Figure 1: The temperature sensitive survival assay shows that the pcspA system with the toxin-antitoxin (ccdA-ccdB) no colonies are present in the plates incubated at temperatures below 37°C compared to the control, pcspA system with reporter gene.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 44
  Illegal PstI site found at 1089
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1089
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 44
  Illegal PstI site found at 1089
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 44
  Illegal PstI site found at 1089
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 985
  

References

1.Stirling, F., Bitzan, L., O'Keefe, S., Redfield, E., Oliver, J., Way, J., & Silver, P. A. (2017). Rational Design of Evolutionarily Stable Microbial Kill Switches. Molecular cell, 68(4), 686–697.e3. https://doi.org/10.1016/j.molcel.2017.10.033