Difference between revisions of "Part:BBa K079031:Experience"
 
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Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated in 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 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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[[Image:TabellaPromotori3.png|center|400px |thumb|Table 1 - Promoter fluorescence ratio after microscope analysis]]
  
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Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated
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The same sample were collected for fluorescence analysis with the Tecan M200 fluorimeter (Table 2) and the fluorescence ratio was confirmed:
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in 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 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 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]]
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[[Image:TabellaPromotoriGrafico2.png|center|400px |thumb|Table 2 - Promoter fluorescence ratio after fluorimeter 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 Tekcan 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 then obtained (OD = 0.1) and cell let to grow a 37 °C in a Tecan 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.
  
 
[[Image:GrowthCurve1.png|center|600px |thumb|Fig.1 - Growth curve]]
 
[[Image:GrowthCurve1.png|center|600px |thumb|Fig.1 - Growth curve]]
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[[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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At the equilibrium once again fluorescence/OD BBa_K079032/ BBa_K079031 ratio was about 1.20 (Fig. 3). A relevant experimental result is the roughly 30fold increase in the fluorescence signal  from the single bacterial cell occurring during the time course. A possible explanation of this observation could rely on the required activation of the major s subunit of RNA polymerase for transcription of most of the genes expressed in the exponential growth phase (Jishage M, Ishihama A. Proc Natl Acad Sci USA 1998; 95: 4953–8. See reference section). Too low fluorescence per cell at the beginning of the monitoring, possibly too close to the lower threshold of the fluorimeter, may also explain why BBa_K079032/ BBa_K079031 ratio was clearly apparent only after 8 hrs in culture.
  
 
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Latest revision as of 01:59, 22 October 2009

Dh5alpha cells transformed with BBa_K079032 and BBa_K079031 were inoculated in 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 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

The same sample were collected for fluorescence analysis with the Tecan M200 fluorimeter (Table 2) and the fluorescence ratio was confirmed:


Table 2 - Promoter fluorescence ratio after fluorimeter analysis

Dilutions from the O/N grown cultures were then obtained (OD = 0.1) and cell let to grow a 37 °C in a Tecan 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 the roughly 30fold increase in the fluorescence signal from the single bacterial cell occurring during the time course. A possible explanation of this observation could rely on the required activation of the major s subunit of RNA polymerase for transcription of most of the genes expressed in the exponential growth phase (Jishage M, Ishihama A. Proc Natl Acad Sci USA 1998; 95: 4953–8. See reference section). Too low fluorescence per cell at the beginning of the monitoring, possibly too close to the lower threshold of the fluorimeter, may also explain why BBa_K079032/ BBa_K079031 ratio was clearly apparent only after 8 hrs in culture.

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