Part:BBa_K1106006:Experience
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Applications of BBa_K1106006
Method A culture of Rhizobium leguminosarum was grown overnight (ON), in TY medium, at 30°C. Besides, a culture of E. coli carrying a plasmid that encodes mRFP under a hybrid promoter inducible by the Lux system of Vibrio fischeri , and repressible by the phage repressor P22 C2 (Bba_K1106000) was also grown ON, at 37°C, in LB with the appropriate antibiotic. As a positive control, a Chromobacterium violaceum culture was grown at 30°C in LB medium. Afterwards, the three cultures were centrifuged. E. coli (DH5α strain) and Chromobacterium violaceum culture’s supernatants were discarded and the pellets were resuspended in Rhizobium leguminosarum culture’s supernatant, containing AHL, which would induce mRFP expression in E. coli, and violacein expression in Chromobacterium violaceum. As a negative control, an "E. coli" culture containing the hybrid promoter was kept in the same medium at which it was previously grown. Cultures were then grown for 70 hours in this conditions, taking aliquots after 0, 7, 24 and 70 hours of induction. Finally, bacteria were sonicated and fluorescence measured, normalized by cell growth (OD 600nm).
Results A greater RFP fluorescence production can be seen in the culture induced with AHL, compared with the negative control. Besides, the culture of Chromobacterium violaceum turned violet 6 hours after Rhizobium leguminosarum conditioned medium was added.
Conclusions
According to the results, the hybrid promoter (Bba_K145150) can be induced by AHL-LuxR. However, fresh medium mRFP curve suggests a high basal transcription from this promoter.
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