Part:BBa_K079031:Experience

Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated in [http://2009.igem.org/Team:Bologna/WetlabProtocols M9 medium] at 8.00 p.m. after O/N growth (about 12 h), samples 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 obtain a significant representation of bacterium fluorescence, it was necessary to acquire several images, each one reporting a sufficient number of bacterial cells. VIFluoR operates the image segmentation and then recognises the bacterial cells yielding the mean fluorescence per bacterium as the output. The final BBa_K079032/ BBa_K079031 fluorescence ratio was equal to 1.20±0.4 (Table 1).

Table 1 - Promoter fluorescence ratio after microscope analysis

The same samples were diluited to an OD equal 0.1 and a growth in time was performed with a Tecan spectrofluorimeter. Both optical density (OD) and fluorescence level were analized for 12 h (Fig.1 and Fig.2, respectively). Fluorescence was then normalized on the OD value (Fig.3).

Fig.1 - Growth curve

Fig.2 - Fluorescence

Fig.3 - Fluorescence curve over OD

As it can be seen from the figures above, data from the fluorimeter analysis agreed with the microscope image analysis. Indeed, the promoter fluorescence ratio was about 1.2. From the figures above, it can be noticed that the fluorescence expression levels are different in the different bacterial phases of growth.

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