Difference between revisions of "Part:BBa K1679011"

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<partinfo>BBa_K1679011 short</partinfo>
 
<partinfo>BBa_K1679011 short</partinfo>
  
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This is a device containing a promoter (BBa_J23106),a RNA thermometer (BBa_K115002), two terminators (BBa_B0015) and a RFP coding sequence (BBa_E0010) on the pSB1C3, which can be easily transformed into E.coli.
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This is a device containing a promoter (BBa_J23106),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.
 
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 42°C, RFP would be expressed in theory.  
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When the temperature rises above 37°C, RFP would be expressed in theory.  
  
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===Usage and Biology===
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===Experiments===
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[[File:OUC-China_project_thermo-regulator_2.png|450px|thumb]]
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[[File:RNAt.jpg|450px|thumb|centre]]  
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[[File:3plateRNAT.png|450px|thumb]]
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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.
 
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.]]
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== Liquid test ==
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[[File:106-002.jpg|px1500|thumb|centre|These are our K16790011 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.]]
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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 works well in liquid LB medium.]]
  
  
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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℃. 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: only FourU functioned, and FourU under the control of promoter J23119 worked best. Our K1679010 doesn't work well as K1679008 and K1679017, but the difference between 37℃ and 42℃ condition can be seen easily.
 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1679011 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K16790011 SequenceAndFeatures</partinfo>

Latest revision as of 02:31, 19 September 2015

promoter+RNA thermometer+RFP

This is a device containing a promoter (BBa_J23106),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

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.

Liquid test

These are our K16790011 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 No part name specified with partinfo tag.