Difference between revisions of "Part:BBa K1679012"

(Created page with "__NOTOC__ <partinfo>BBa_K1679012 short</partinfo> This is a device containing a promoter (BBa_J23106),a RBS (BBa_K115003) and a RFP coding sequence (BBa_E0010) on the pSB1C3, wh...")
 
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<partinfo>BBa_K1679012 short</partinfo>
 
<partinfo>BBa_K1679012 short</partinfo>
  
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This is a device containing a promoter (BBa_J23106),a RBS (BBa_K115003) and a RFP coding sequence (BBa_E0010) on the pSB1C3, which can be easily transformed into E.coli.In theory,When the temperature rises above 37°C,It can produce RFP.However,according to our experimental,It can't appear red.
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This is a device containing a promoter (BBa_J23106),a RNA thermometer (BBa_K115003) and a RFP coding sequence (BBa_E0010) on the pSB1C3, which can be easily transformed into E.coli.
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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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When the temperature rises above 37°C, RFP would be expressed in theory.  
  
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===Experiments===
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[[File:RNAt.jpg|450px|thumb|centre]]
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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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== Agar plate test ==
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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: FourU worked best under all these promoters and show great switch activity when be used cooperatively with J23101 and J23119. Our K1679012 didn't show significant difference between 37℃ and 42℃ conditions.]]
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== Liquid test ==
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[[File:106-003.jpg|px1500|thumb|centre|These are our K1679012 which were cultured at 28℃, 35℃, 37℃, 40℃, and 42℃ from right to left. It didn't show significant difference between these temperature in liquid LB medium.]]
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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).
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[[File:RNAT total.png|800px|thumb|left|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 dosen't work well in liquid LB medium.]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1679012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1679012 SequenceAndFeatures</partinfo>

Latest revision as of 02:32, 19 September 2015

promoter+RNA thermometer+RFP

This is a device containing a promoter (BBa_J23106),a RNA thermometer (BBa_K115003) 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

RNAt.jpg

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. Our K1679012 didn't show significant difference between 37℃ and 42℃ conditions.

Liquid test

These are our K1679012 which were cultured at 28℃, 35℃, 37℃, 40℃, and 42℃ from right to left. It didn't show significant difference 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 dosen't work 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 713
    Illegal AgeI site found at 825
  • 1000
    COMPATIBLE WITH RFC[1000]