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Team:Buenos Aires/ hybrid
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'''Method''' | '''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 HSL, which would induce mRFP expression in E. coli, and violacein expression in Chromobacterium violaceum. | 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 HSL, which would induce mRFP expression in E. coli, and violacein expression in Chromobacterium violaceum. | ||
Revision as of 23:29, 27 September 2013
Hybrid promoter induction and repression
Hybrid promoter induction
Objective
Characterize the response of the hybrid promoter to the HSL/LuxR inducer complex.
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 HSL, which would induce mRFP expression in E. coli, and violacein expression in Chromobacterium violaceum.
Cultures were then grown for 72 hours, and aliquots were taken at 0, 24 and 72 hours of induction. Finally, bacteria were sonicated and fluorescence measured, normalized by cell growth (OD 600nm).
Results
According to the results shown in the following figure, the hybrid promoter (Bba_K145150) can be induced by HSL-LuxR. However, fresh medium mRFP curve suggests a high basal transcription from this promoter
