Measurement

Part:BBa_K669006:Design

Designed by: Silvia J Caas   Group: iGEM11_Colombia   (2011-10-28)
Revision as of 14:22, 3 November 2011 by Sylvita1015 (Talk | contribs) (→‎Design Notes)

Allows the measurement of the CBP promoter activity


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 119
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 119
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 119
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 119
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

mCherry protein is used from a BioBrick

We made a construct where the CBP promoter leads the expression of the mCherry reporter. Chitooligosaccharide-Binding Protein (CBP) is a cytoplasmatic protein that regulates the transcriptional activity of the ChiS sensor. When chitin is present in the external media, CBP binds with GlcNAc oligomers (chitin degradation products) and liberates the sensor for the up-regulation of the chitinolitic degradation cascade in Vibrio fischeri ES114. We isolated the promoter of CBP (Part BBa_K669003) and then we built a vector were the fluorofore mCherry (from part BBa_J06702) is under the control of CBP promoter (Part BBa_K669006) in order to characterize CBP promoter transcriptional activity.

The characterization was performed using a fluorometer. We selected the standard part BBa_J23119, a constitutive promoter of E. coli, as a control in our experiments. The strain with the BBa_K669006 and BBa_J23119 were grown in LB media. The experiment was performed at 37 C for 600 seconds. As the part BBa_J23119 carries the fluorofore mRFP1 and our part BBa_K669006 carries mCherry, it was necessary to consider the differences between the two fluororores in order to be able to compare the fluorescence intensities.


BBa K669006 1.jpg


Were Brigthness was calculated using the formula Brigthness = Extinction coefficient * Quantum yield / 1000

The intensities were normalized to delete the blank autofluorescence and also the difference between the brightness of each fluorescence protein (Brightness of mRFP1 = 12,5 and Brightness of mCherry = 15,88). As the experiment was performed at 37 C, the strains continued growing during the experiment. For that reason the final OD was measured and the difference in OD was used to normalize the data (both strains were in exponential growth).

Source

Vibrio fischeri ES114

References