Part:BBa_K1686033:Experience

Applications of BBa_K1686033

To verify that the OsmY promoter is only active in stationary phase, we realized Curdlan quantitative analyses every hour of a culture in LB medium. We could therefore show a parallel between growth and Curdlan production. The culture temperature was changed when stationary phase was obtained.

→ As we can see, Curdlan appears after the switch at 27°C. So, OsmY promoter is active in stationary phase only.

Fig.1: OsmY promoter characterization : The production of Curdlan was made in LB medium at 37°C during exponential phase and after a temperature switch at 27°C at the beginning of the stationary phase (to slow down cell metabolism). The Curdlan concentration was calculated with absorption measures (excitation wavelength at 398nm and emission wavelength= 502nm) after Curdlan purification from cells. We used aniline blue dye since it is specific of beta glucans.

We tested the Curdlan produced by this part by spraying a 30 mg/L solution onto vine leaves. After one day we infected those leaves with Mildew spores and let the infection develop for 6 days.

The leaves sprayed with our Coli derived Curdlan showed a 85% reduction of spore developpment compared to the mock sprayed with water.

A control with commercial Curdlan showed 95% reduction but a higher rate of necrosis than our product. Necrosis is the result of overactivation of the plant defense system. Further tests would be needed to find the concentration maximizing protection with minimal necrosis.

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