Difference between revisions of "Part:BBa K1679009"
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This is a device containing a promoter (BBa_J23101),a RNA thermometer (BBa_K115003) and a RFP coding sequence (BBa_E0010) on the pSB1C3, which can be easily transformed into E.coli. | This is a device containing a promoter (BBa_J23101),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; | + | 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. | When the temperature rises above 37°C, RFP would be expressed in theory. | ||
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== Agar plate test == | == Agar plate test == | ||
| − | [[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 | + | [[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 K1679009 didn't show significant difference between 37℃ and 42℃ conditions.]] |
== Liquid test == | == Liquid test == | ||
| − | [[File: | + | [[File:101-003.jpg|px1500|thumb|centre|These are our K1679009 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). | 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). | ||
[[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.]] | [[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.]] | ||
Latest revision as of 02:29, 19 September 2015
promoter+RNA thermometer+RFP
This is a device containing a promoter (BBa_J23101),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
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
Liquid test
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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 713
Illegal AgeI site found at 825 - 1000COMPATIBLE WITH RFC[1000]