Difference between revisions of "Part:BBa K1679014"

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<partinfo>BBa_K1679014 short</partinfo>
 
<partinfo>BBa_K1679014 short</partinfo>
  
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This is a device containing a RBS (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.
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This is a device containing a RBS (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, this RNA terminator would turn on and RFP would be expressed in theory.
 
When the temperature rises above 37°C, this RNA terminator would turn on and RFP would be expressed in theory.
  
 
===Usage and Biology===
 
===Usage and Biology===
 
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.
 
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.
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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.
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[[File:3plateRNAT.png|450px|thumb|centre| 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: this RNA thermometer worked best under all these promoters and show great switch activity when be used cooperatively with J23101 and J23109. ]]
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[[File:RNAT total.png|800px|thumb|left|After 36 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 solid LB medium also.]]
  
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[[File:3plateRNAT.png|450px|thumb|left| 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: this RNA thermometer worked best under all these promoters and show great switch activity when be used cooperatively with J23101 and J23109. ]]
 
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1679014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1679014 SequenceAndFeatures</partinfo>

Latest revision as of 01:28, 19 September 2015

RNA thermometer (FourU) +mRFP

This is a device containing a RBS (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, this RNA terminator would turn on and RFP would be expressed in theory.

Usage and Biology

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.

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: this RNA thermometer worked best under all these promoters and show great switch activity when be used cooperatively with J23101 and J23109.
After 36 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 solid LB medium also.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 613
    Illegal AgeI site found at 725
  • 1000
    COMPATIBLE WITH RFC[1000]