Part:BBa_K4345020

5' UTR of hsp17 with ccdA fused to sfGFP

Design of a thermoregulated translation of antitoxin ccdA. Fluorescent protein sfGFP was linked to the antitoxin with a rigid linker for visualisation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1149
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 569
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The figure below shows the secondary structure of the RNA thermometer.

Image obtained from Kortmann et al., 2010, edited with biorender and UNAfold.

Results

Below you can find an example of the results found after the induction tests of the RNA thermometer.

Two 96 well plates were prepared with 200 µL of the relevant selection medium (LB + 100 µg/mL ampicillin) and the plates were covered with a breathable film. The cultures E. coli DH5α were diluted 50 times in these well plates and grown at 25°C or 37°C overnight at 180 rpm. The next day, both the OD₆₀₀ as the fluorescence intensity were measured with the CLARIOstar (BMG Labtech, Ortenberg, Germany). Blancs (LB) and negative controls (culture with pLO_SNAP plasmid) are subjected to the same protocol. All measurements were performed in triplicate. To visualise the results for this picture, the successful cultures were grown on plates at the different temperatures.

In the figure below, the construct transformed into different cell types: BL21 (top) and K12 M1665 (bottom).

References

Kortmann, J., Sczodrok, S., Rinnenthal, J., Schwalbe, H., & Narberhaus, F. (2010, December 3). Translation on demand by a simple RNA-based thermosensor. Nucleic Acids Research, 39(7), 2855–2868. https://doi.org/10.1093/nar/gkq1252