Difference between revisions of "Part:BBa K079032:Experience"

Latest revision as of 01:55, 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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@@ Line 4: / Line 4: @@
 [[Image:TabellaPromotori3.png|center|400px |thumb|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 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.
+The same sample were collected for fluorescence analysis with the Tecan M200 fluorimeter (Table 2) and the fluorescence ratio was confirmed:
+[[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]]
@@ Line 10: / Line 15: @@
 [[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 that this value becames clearly apparent only at an advanced stage of bacterial cell growth, i.e. after 8 hrs. This may depend on the roughly tenfold increase in the fluorescence signal  from the single bacterial cell occurring during the same time span. In fact, a low fluorescence per cell at the beginning of the monitoring, possibly too close to the lower threshold of the fluorimeter, may explain why this difference could be non apparent at that time. This observation consistently raises a question about how to explain this observed increase in fluorescence. A possible explanation could rely on the 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 the reference section).
+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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