Difference between revisions of "Part:BBa K079031:Experience"

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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-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).
+<div style="text-align:justify">
+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).
 [[Image:TabellaPromotori3.png|center|400px |thumb|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:
-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.
+[[Image:TabellaPromotoriGrafico2.png|center|400px |thumb|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.
 [[Image:GrowthCurve1.png|center|600px |thumb|Fig.1 - Growth curve]]
 [[Image:FluorescenceCurveAbsolute1.png|center|600px |thumb|Fig.2 - Fluorescence]]
 [[Image:FluorescenceCurveOverOD1.png|center|600px |thumb|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.
-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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