Part:BBa_K606035
pT7 SpoVG GFP T7terminator.
T7 RNA polymease controlled GFP expression system for B. subtilis and E. coli
Usage and Biology
This part is the concatenation of the promoter pT7 (I719004) with the RBS SpoVG (K143021), that is design for B. subtilis but that also works in E. coli. This part works very efficiently and have been caracterized in E. coli BL21 cells. The part is in pSB1C3.
See the experiment page of this part or our wiki page[http://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion] for additional information
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 917
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 693
Characterization
In order to characterize the pT7 promotor, we used the construct: pT7-RBS-GFP-T7ter. This construct was transformed into BL21 strains expressing the T7 polymerase under IPTG induction.
Fluorescence kinetics
The measurements have been carried out on a spectrophotometer, at 37°C under transient shaking. The experiment lasted 4h, we tested several colonies and several IPTG concentrations. The OD 600nm and the fluorescence of the GFP (exc: 470nm / meas:515 nm) was measured every 5 min, and the ratio of the two was calculated.
All values were normalized by substracting the fluorescence/OD value of the well with 0 mM IPTG at time 0. The values given are in arbitrary units.
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After 2 hrs of induction, we see a clear increase of the fluorescence proportional to the IPTG concentration (that is to say with the quantity of T7 polymerase induced in the cell). After 4 hrs, the expression of GFP under the pT7 is still not saturated
Here, we plot the ratio of induction of the T7 polymerase dependant construct for the different concentrations of IPTG at a given time (4 hrs), taking the well with 0 IPTG at time 0 as the reference.
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