Difference between revisions of "Part:BBa K606035"

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===Characterization===
 
===Characterization===
  
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Growth of this part in BL21 strain under various concentration of IPTG.
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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.
  
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<h3>Growth</h3>
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<h3>Fluorescence kinetics</h3>
  
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<p>The measurements have been carried out on a spectrophotometer, at 37°C under transient shaking, for 4h, for several colonies and several range of IPTG concentration. 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.</p>
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<p>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.</p>
  
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<p>All values were normalized by subtracting the fluorenscence/OD value of the well with 0 mM IPTG at time 0. The values given are in arbitrary units.</p>
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<p>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.</p>
  
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</html>
 
[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
 
[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
 
<html>
 
<html>
  
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<p>First, we see an inflexion in the curve that is due to the stong influence of the IPTG on the metabolism of the cells. Then, the bacteria start growing again. We see a clear increase of the fluorescence with the IPTG concentration, that is to say with the quantity of T7 polymerase in the cell. All values were normalized by subtracting the fluorenscence/OD value of the well with 0 mM IPTG at time 0</p>
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<p>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</p>
  
 
</html>
 
</html>
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription saturated and non saturated cells]]
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription]]
 
<html>
 
<html>
  
 
<p>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.</p>
 
<p>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.</p>

Revision as of 00:34, 22 September 2011

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 917
  • 1000
    INCOMPATIBLE 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.

</html>

Fig1: Growth curves for BL21 strain carrying the part

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

Fig2: Comparison of the Fluo/OD ratio for transcription

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.