Difference between revisions of "Part:BBa K606035"

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===Usage and Biology===
 
===Usage and Biology===
  
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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.
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This part is the concatenation of the promoter pT7 (I719004) with the RBS SpoVG (K143021), that design is for B. subtilis 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
 
See the experiment page of this part or our wiki page[http://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion] for additional information
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K606035 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K606035 SequenceAndFeatures</partinfo>
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===Characterization===
 
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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>Fluorescence kinetics</h3>
 
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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 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>
 
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[[Image:GrowthpT7GFPt7ter.png|center|thumb|500px|Fig1: Growth curves for BL21 strain carrying the part]]
 
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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>
 
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</html>
 
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[[Image:pT7GFPSaturated.png|center|thumb|600px|Fig2: Comparison of the Fluo/OD ratio for transcription]]
 
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<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>
 

Latest revision as of 18:58, 23 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 design is for B. subtilis 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