Difference between revisions of "Part:BBa K079032:Experience"
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Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated  
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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).
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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.
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The final BBa_K079032/ BBa_K079031 fluorescence ratio was equal to 1.20±0.4 (Table 1).
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[[Image:TabellaPromotori3.png|center|400px |thumb|Table 1 - Promoter fluorescence ratio after microscope analysis]]
 
[[Image:TabellaPromotori3.png|center|400px |thumb|Table 1 - Promoter fluorescence ratio after microscope analysis]]
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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).
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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.
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[[Image:GrowthCurve1.png|center|600px |thumb|Fig.1 - Growth curve]]
 
[[Image:GrowthCurve1.png|center|600px |thumb|Fig.1 - Growth curve]]
 
[[Image:FluorescenceCurveAbsolute1.png|center|600px |thumb|Fig.2 - Fluorescence]]
 
[[Image:FluorescenceCurveAbsolute1.png|center|600px |thumb|Fig.2 - Fluorescence]]
 
[[Image:FluorescenceCurveOverOD1.png|center|600px |thumb|Fig.3 - Fluorescence curve over OD]]
 
[[Image:FluorescenceCurveOverOD1.png|center|600px |thumb|Fig.3 - Fluorescence curve over OD]]
  
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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.
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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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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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Revision as of 23:51, 21 October 2009

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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