Part:BBa_K4244023

placUV5:ccdA + pcspA:ccdB

Disclaimer: this composite part is supposed to be compatible with RFC[1000]. 
In fetching the separate part an unknown error occurs resulting in a BsaI site in the ccdB part. 
For a correct, RFC[1000] compatible part, see BBa_K4244099

BBa_K4244099

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 control of 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 temperatures cspA stabilizes and ccdB overexpresses ccdA, which leads to cell death.

This construct has been transformed in E. coli Nissle 1917 and tested with a temperature sensitive survival assay. This experiment has been carried out by plating overnight culture grown at 37°C in different agar plates, which are incubated at 37°C (control), 30°C, 25°C and 20°C. The result of this experiment, shown in Figure 1, allows to say that the pcspA with the toxin-antitoxin system works as there is no colony present at lower temperature than 37°C.

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