Difference between revisions of "Part:BBa K1679007"

Revision as of 20:13, 18 September 2015

promoter+RNA thermometer+RFP

This is a device containing a promoter (BBa_J23101),a RNA thermometer (BBa_K115001) 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 42°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 K1679007 didn't show significant difference between 37℃ and 42℃ conditions.

Liquid test

These are our K1679007 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 works 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 700
Illegal AgeI site found at 812
1000
COMPATIBLE WITH RFC[1000]

@@ Line 18: / Line 18: @@
 [[File:101-001.jpg|px1500|thumb|centre|These are our K1679007 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 36 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 works well in liquid LB medium.]]