Part:BBa_K1140006

pTet + 37 oC RNA thermometer + mCherry (LVA)

Transcription of mCherry [1] is under the regulation of a pTet promoter, based on [2,3]. The promoter is constitutively ON and repressed by TetR transcription factor. The TetR repression is inhibited by the addition of tetracycline or its analog aTc to the media. Translation is also regulated by a synthetic RNA thermometer [4] that allows translation only over 37°C (figure 1).

Figure 1. Predicted secondary structure of the synthetic RNATs contained in this part, as calculated by Mfold. The orange rectangles highlight nucleotides belonging to the SD sequence.

Usage and Biology

RNA thermometers (RNAT) are temperature sensing RNA sequences that regulate translation by preventing the ribosome from binding the transcript until higher temperatures shift it to an open structure. Several naturally occurring RNA thermometers have been described and synthetic sequences that emulate them have been designed and proved to regulate genetic expression at different temperature ranges. The RNAT herein implemented, designed and dubbed u6 by Neupert et al. [4], is a synthetic sequence that allows translation only at temperatures over 37 °C (figure 2, 3). This part can be incorporated into a genetic circuit to integrate transcriptional and post-transcriptional regulation, through Tet Repressor and u6-RNAT, respectively.

Figure 2. Temperature dependence of mCherry translation by u6 RNA thermometer in E. coli. Tet repressor is NOT present in this test. Two control cultures without mCherry sequence are included for growth and color comparison. a) Control 30ºC b) Control 37ºC c) 30ºC d) 37ºC.

Figure 3. Average relative fluorescence values of cultures carrying the construction, incubated at 31 and 37ºC.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 63
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 676
  Illegal AgeI site found at 788
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

direction	Forward
function	Integrated Transcriptional and Translational regulation
protein	mCherry
switch_point	37 celsius degrees
tag	LVA