Part:BBa_K3788017:Experience
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Applications of BBa_K3788017
Methods
In order to characterize the promoter strength, we fused it to the GFP encoding sequence BBa_E0040 and to the timer-lysis device part BBa_K3788018 (composite part BBa_K3788019). Induction of the SOS response was induced by mitomycin C treatments and the fluorescence was recorded in a dose-dependent manner using a microplate reader. Following iGEM Measurement recommendations, the GFP fluorescence intensity was converted to a concentration of fluorescein protein thanks to a standard curve of fluorescein dilution series.
Results
The cells carrying the BBa_K3788019 construct were grown with various concentrations of mitomycin, ranging from 0 to 800 ng.ml-1. The OD (600 nm) and the amount of green fluorescence were recorded over time.
Our data confirmed that the promoter part BBa_K3788017 responds to mitomycin in a dose dependent manner from 50 to 800 ng.ml-1. The signal is significantly detected 90 min to 130 min after the beginning of the experiment, depending of the inducer tested concentration.
Moreover, we showed that the promoter is strongly repressed in the absence of the inducer, as no fluorescence signal is detected for the untreated control during the time course of the experiment (450 min).
Finally, we demonstrated that during the 90 to 210 min window after induction, there is a linear relationship between the accumulation of GFP signal and Mitomycin concentration. We established titration curves for this time period (R²>0.975).
We concluded that BBa_K3788017 is a new and strongly regulated promoter that might be of great interest to the synthetic biology community.
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