Difference between revisions of "Part:BBa K2033000:Design"
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<div style="text-align: center;">[[File:T--Arizona State--AUBRFP.png]]</div>
 
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Mass spectrometry was also used to characterize AubI. After purification of the AHL serum by HPLC, a matrix was combined with the sample and run using mass spectrometry. The comparison below between the negative control and the sample shows that a peak around 283.9 m/z appeared in the sample, which matches the predicted mass to charge ratio of the AubI AHL.
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<div style="text-align: center;">[[File:T--Arizona_State--Aubmassspec.png]]</div>
 
===Source===
 
===Source===
 
This synthase was recovered from a metagenomic project on a soil sample. The specific source is unknown.
 
This synthase was recovered from a metagenomic project on a soil sample. The specific source is unknown.

Revision as of 19:38, 12 October 2016


N-dodecanoyl-L-homoserine lactone (C(12)-HSL) Sender- AubI


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 535
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 510
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The original AubI part was sampled from soil and is thought to be from an unknown soil-borne bacteria. The bacteria was transformed into BL21 Competent E. coli cells to produce sufficient stock for future experiments. Of the 70 colonies that were produced, three samples were taken for verification. After mini-prepping, the gel verification affirmed the identity of the sample, as shown below:

T--Arizona State--Gel4.jpg
Lanes 3,4,5 correspond to AubI samples; Lane 1 contains the kb+ ladder.

After gel verification and sequencing, the AubI part was retransformed in BL21(DE3) E. coli and run in a 96-well plate from 580-610nm to measure mCherry production. This produced the curve below, indicating that the AHL is being produced by the sender, since mCherry production increased over an 8hour read time. This is because both the AHL and mCherry genes are found on the Sender.

T--Arizona State--AUBRFP.png

Mass spectrometry was also used to characterize AubI. After purification of the AHL serum by HPLC, a matrix was combined with the sample and run using mass spectrometry. The comparison below between the negative control and the sample shows that a peak around 283.9 m/z appeared in the sample, which matches the predicted mass to charge ratio of the AubI AHL.

T--Arizona State--Aubmassspec.png

Source

This synthase was recovered from a metagenomic project on a soil sample. The specific source is unknown.

References

(1) Nasuno, E., N. Kimura, M. J. Fujita, C. H. Nakatsu, Y. Kamagata, and S. Hanada. "Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach." Applied and Environmental Microbiology 78.22 (2012): 8067-074. Web