Part:BBa_K079032:Experience

Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated in [http://2009.igem.org/Team:Bologna/WetlabProtocols M9 medium] O/N. The day after, samples of bacterial cells in the stationary phase were collected and slide prepared for image acquisition with the optical microscope. Images were then analyzed with the [http://2009.igem.org/Team:Bologna/Software VIFluoR] software to analyse bacterial fluorescence. Mean fluorescence per bacterium was 51.3± 8.3 a.u. for BBa_K079032 and 43.7±10.4 a.u. for BBa_K079031. Fluorescence ratio BBa_K079032/ BBa_K079031 was 1.20±0.4 (Table 1).

Table 1 - Promoter fluorescence ratio after microscope analysis

Dilutions from the O/N grown cultures were obtained (OD = 0.1) and cell let to grow a 37 °C in a Tecan M200 spectrofluorimeter. Both optical density (OD; Fig. 1) and fluorescence level (Fig. 2) were analized during 12 h. Fluorescence/OD ratio is shown over time in Fig. 3.

Fig.1 - Growth curve

Fig.2 - Fluorescence

Fig.3 - Fluorescence curve over OD

At the equilibrium once again fluorescence/OD BBa_K079032/ BBa_K079031 ratio was about 1.20 (Fig. 3). A relevant experimental result is that this proportion was not constant all over the entire time course of the experiment, but became apparent only at an advanced stage of bacterial cell growth.

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