Part:BBa_K1679017

Promoter+RNA thermometer (FourU) +mRFP

This is a device containing a promoter (BBa_J23119),a RNA thermometer (BBa_K115002) and a RFP coding sequence (BBa_E0010) on the pSB1C3, which can be easily transformed into E.coli. RNA Thermometers(RNAT) are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, RNAT folds into structure, blocking access of ribosome; At high temperature, RNAT switch from off to open conformation, increasing the efficiency of translation initiation. When the temperature rises above 37°C, RFP would be expressed in theory.

Experiments

TUDelft-2008 has modified 3 kinds of RNAT: ROSE(BBa_K115001), FourU(BBa_ K115002), PrfA(BBa_K115003). We constructed each RNAT under the control of 3 different constitutive promoters:BBa_J23101, BBa_J23106, BBa_J23119 and use RFP as a reporter. After construction, the 9 circuits were transformed into DH5α. According to the results of TUDelft-2008, the temperature threshold is 37℃ for FourU and PfrA, 42℃ for ROSE. Thus, we set the culture temperature to 28℃, 37℃ and 42℃ on solid LB medium while 28℃, 35℃, 37℃, 40℃, and 42℃ in liquid LB medium.

Agar plate test

For one temperature, we spotted one strain on 3 different plates, each plate contains 9 different strains. After 48 hours, as we can see in the photo: FourU worked best under all these promoters and show great switch activity when be used cooperatively with J23101 and J23119.

Liquid test

These are our K1679017 which were cultured at 28℃, 35℃, 37℃, 40℃, and 42℃ from right to left. It has significant differences between these temperature in liquid LB medium.

The 9 circuits were tested through plate reader as well. We set 5 temperature: 28℃, 35℃, 37℃, 40℃ and 42℃. After 41 h, when some replicates had changed color, we calculated the Fluorescence(excitation wavelength-584 nm and emission wavelength-607 nm) and OD(600).

After 41 h culturing in solid LB medium , when some replicates had changed color, we calculated the Fluorescence(excitation wavelength-584 nm and emission wavelength-607 nm) and OD(600).This part works well in liquid LB medium.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 656
  Illegal AgeI site found at 768
  
• 1000
  COMPATIBLE WITH RFC[1000]