Difference between revisions of "Part:BBa K1679018"

Latest revision as of 02:20, 19 September 2015

Promoter+RNA thermometer (PrfA) +mRFP

This is a device containing a promoter (BBa_J23119),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

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 K1679018 didn't show significant difference between 37℃ and 42℃ conditions.

Liquid test

These are our K1679018 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]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1679018 short</partinfo>
-This is a device containing a promoter (BBa_J23109),a RNA thermometer (BBa_K115003),two terminators (BBa_B0015) 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_J23119),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.
-===Usage and Biology===
+===Experiments===
-[[File:OUC-China_project_thermo-regulator_2.png|450px|thumb]]
+[[File:RNAt.jpg|450px|thumb|centre]]
-[[File:3plateRNAT.png|450px|thumb]]
 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 ==
+[[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 K1679018 didn't show significant difference between 37℃ and 42℃ conditions.]]
+== Liquid test ==
-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 K1679018 didn't show significant difference between 28℃ and 37℃ conditions.
+[[File:119-003.jpg|px1500|thumb|centre|These are our K1679018 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).
+[[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>
 <partinfo>BBa_K1679018 SequenceAndFeatures</partinfo>