Part:BBa_K4761239

report-U9(new)

Temperature-sensitive reporter unit U9 for pET28a vector. Activates at around 37℃.

We inserted the RNA thermometer element into the plasmid encoding the EGFP gene by enzyme cleavage and enzyme link, and the specific position was between the promoter and the start codon to obtain the combined part REPORT U9(new)[1][2]. Through the construction of this combination part, we can use the EGFP gene expression strength to characterize the success of the RNA thermometer. The strains transferred to the plasmid were cultured at different temperatures, and the expression intensity of EGFP was obtained at different temperatures, which reflected the opening temperature of the RNA thermometer.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 4
  Illegal SpeI site found at 23
  Illegal PstI site found at 29
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 23
  Illegal PstI site found at 29
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 43
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 4
  Illegal SpeI site found at 23
  Illegal PstI site found at 29
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 4
  Illegal SpeI site found at 23
  Illegal PstI site found at 29
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 15
  

Result=

The strains were first cultured in vitro, and after a certain period of culture, 2mL was taken into a small centrifuge tube, and the fluorescence intensity of the discarded supernatant was observed by naked eye to reflect the construction of the RNA thermometer, as shown in Figure 1.

Fig.1 A quick comparison of RNA thermometer constructs by temperature-controlled EGFP expression in E. coli

It can be seen that the expression of the RNA thermometer element is turned on at 30,37℃ and not expressed at 17,22 ℃.

References

[1]Neupert, J., Bock, R. Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria. Nat Protoc 4, 1262–1273 (2009). [2]Juliane Neupert, Daniel Karcher, Ralph Bock, Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli, Nucleic Acids Research, Volume 36, Issue 19, 1 November 2008, Page e124, https://doi.org/10.1093/nar/gkn545