Difference between revisions of "Part:BBa K669006:Design"

 
 
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<partinfo>BBa_K669006 short</partinfo>
 
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===Design Notes===
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Chitooligosaccharide-Binding Protein (CBP) is a cytoplasmatic protein that regulates the transcriptional activity of the ChiS sensor.
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mCherry protein is used from a BioBrick
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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.
 +
 
 +
[[Image:BBa_K669006_1.jpg]]
 +
 
 +
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.
 +
 
 +
[[Image:BBa_K669006_2.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).
 +
 
 +
[[Image:BBa_K669006_3.jpg]]
 +
 
 +
The resultant data is summarized in the above graphic.
 +
As you can see, our system to measure the expression of CBP promoter works properly (Our bacteria is expressing mCherry!!!) and it appears that its expression is at strong when compared with the expression of the strongest member of the family of constitutive promoters of E. coli (BBa_J23119)
 +
 
 +
===Epifluorescence Microscopy===
  
 +
[[Image:BBa_K669006_4.jpg|800px]]
  
 +
Our bacteria is expressing mCherry!!!
  
 
===Source===
 
===Source===
  
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Vibrio fischeri
+
Vibrio fischeri ES114
  
 
===References===
 
===References===

Latest revision as of 15:34, 3 November 2011

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]


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.

BBa K669006 1.jpg

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 2.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).

BBa K669006 3.jpg

The resultant data is summarized in the above graphic. As you can see, our system to measure the expression of CBP promoter works properly (Our bacteria is expressing mCherry!!!) and it appears that its expression is at strong when compared with the expression of the strongest member of the family of constitutive promoters of E. coli (BBa_J23119)

Epifluorescence Microscopy

BBa K669006 4.jpg

Our bacteria is expressing mCherry!!!

Source

Vibrio fischeri ES114

References