Part:BBa_K4761230

report-U6-1

Temperature-sensitive reporter unit U6-1. Activates at 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 U6-1[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 EcoRI site found at 1
  Illegal SpeI site found at 15
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal SpeI site found at 15
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1
  Illegal SpeI site found at 15
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1
  Illegal SpeI site found at 15
  
• 1000
  COMPATIBLE WITH RFC[1000]

Result

The strains were first cultured in vitro, and then 24-well plates were added to ensure that OD values were the same. Each sample was repeated three times and cultured at different temperatures. After that, fluorescence values were measured by enzymoleter and OD values were measured by spectrophotometer. Finally, RFU/OD values were obtained to characterize fluorescence intensity per unit concentration,as shown in Table 1.

Table.1 RFU/OD-Temperature,U0:No RNA thermometer.

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