Part:BBa_K1529797:Experience
Plux_CmR_RhlI
Contents
Materials and Methods
-Strain
All the samples were JM2.300 strain.
3OC12HSL-dependent CmR expression Protocol
1.Construction
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-GFP-Ptet-RhlR (psB6A1), PlacIq-CmR (pSB3K3) (Positive control)
2.Assay protocol
1.Prepare the overnight culture of cell A and B at 37°C.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture)
3. Add 30 microL of suspension in the following medium.
1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)
2) 3 mL of LB containing Amp and Kan + 30 microL DMSO
3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)
4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)
3OC12HSL-dependent C4HSL production Protocol
1.Construction
Sender
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control
Reporter
C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control
E. Ptet-RhlR(pSB6A1), Promoter-less-GFP(pSB3K3)...Negative control
2.Assay protocol
Results
3OC12HSL-dependent CmR expression Result
We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)
Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.
Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.
On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL.
Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol.
As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.
3OC12HSL-dependent C4HSL production Result
For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki].
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