Generator
pT7 GFP T7

Part:BBa_K606035

Designed by: Axel Séguret   Group: iGEM11_Paris_Bettencourt   (2011-09-18)
Revision as of 23:03, 21 September 2011 by Danyel.lee90 (Talk | contribs) (→‎Characterization)

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

Growth of this part in BL21 strain under various concentration of IPTG.

Growth

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.

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.

Fig1: Growth curves for BL21 strain carrying the part

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

Fig2: Comparison of the Fluo/OD ratio for transcription saturated and non saturated cells

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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Categories
Parameters
None