Part:BBa_K1824563

RNA Thermometer U6 (Specially designed for pBAD)

This is a specially designed U6 RNA thermometer that with a unique spacer at the front, which would make it compatible with promoter pBAD BBa_I13453.It is important to noted that this part is not compatible with RFC[10]. XJTLU-CHINA used Gibson method to assembly this part to others.

U6 RNA thermometer have the hairpin structure that harbors the Shine-Dalgarno sequence (SD sequence) and, in this way, make it inaccessible to the 30S unit of the bacterial ribosome, resulting in translational inactivation (Figure 2). The melting temperature of this RNA thermometer is 37 Celsius degree. Once reaching the melting temperature, hairpin structure would vanish and as a result, exposing the SD sequence to trigger the translation process.

Different promoters have their own transcription start sites and, in most cases, + 1 sites are embedded in promoter sequence. Hence, it is normal that transcribed RNA usually carry part of promoter sequence. However, for regulatory parts like RNA thermometer, truncation or alteration of the RNA sequence could be destructive. Hence, special designed RNA spacer between transcribed part of promoters and RNA thermometers are important for maintaining the secondary structure of RNA thermometer.

For pBAD BBa_I13453, transcription starts at TCTCCATA (transcription start site indicated in bold). Based on this, BBa_K1824563 was specially designed with a spacer that had less probability to interact with the functional structure of RNA thermometer.

The possible secondary structure of U6 was simulated by RNAstructure (Fig.1). For testing results of pBAD-U6, See BBa_K1824005.

Left picture: XJTLU-CHINA (2015) Figure 1: Possible secondary structure of RNAT U6.
Right picture: TuDelft (2008) Figure 2: Responsiveness of mRNA structures to environmental cues.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 23
  
• 12
  INCOMPATIBLE WITH RFC[12]

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

  Illegal BamHI site found at 9
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 23
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 23
  
• 1000
  COMPATIBLE WITH RFC[1000]