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Team:UNIPV-Pavia/Methods Materials/Fluorescence
From 2009.igem.org
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| - | == <html><font class="dayw_style">Fluorescence and | + | == <html><font class="dayw_style">Fluorescence and dilutions in water</font></html> == |
| - | The proportionality between fluorescence measurements (GFP and RFP) and different | + | The proportionality between fluorescence measurements (GFP and RFP) and different dilutions of LB was verified in two volumetric dilutions. |
| - | This considerations has not to be read together with the results of the non-liner relationship between fluorescence and water dispensation, because even if different amounts of water in the well means different | + | This considerations has not to be read together with the results of the non-liner relationship between fluorescence and water dispensation, because even if different amounts of water in the well means different dilution of the colture, in that simulation we didn't work with costant volume in the well, as in this case we do. |
The first two measurements must not be taken into account because of the high measure's rumour. | The first two measurements must not be taken into account because of the high measure's rumour. | ||
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| - | [[Image: | + | [[Image:Unipv_fu_dilution_LB.png|500px|Fluorescence and water dilution|thumb|center|Fluorescence (GFP) and water dilution, LB]] |
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[[Image:Unipv_fu_volume_LB.jpg| 500px|Fluorescence and volume|thumb|center|Fluorescence and volume, LB]] | [[Image:Unipv_fu_volume_LB.jpg| 500px|Fluorescence and volume|thumb|center|Fluorescence and volume, LB]] | ||
| - | == <html><font class="dayw_style">Fluorescence and | + | == <html><font class="dayw_style">Fluorescence and dilutions in liquid growing medium</font></html> == |
This experiment was done in two different growing mediums, LB and M9, and in three different total volumes in the well, 100μl, 200μl and 300μl; a black plate was also used to repeat the experiment in LB. | This experiment was done in two different growing mediums, LB and M9, and in three different total volumes in the well, 100μl, 200μl and 300μl; a black plate was also used to repeat the experiment in LB. | ||
| - | The hypothesis of proportionality between fluorescence measurement and colture's | + | The hypothesis of proportionality between fluorescence measurement and colture's dilutions was not verified. |
We observed a non-linear relationship between bacterial quantity in the well and fluorescence (both GFP and RFP) measures, in the two cases, and a non-negligible reliance on the total volume in the well. | We observed a non-linear relationship between bacterial quantity in the well and fluorescence (both GFP and RFP) measures, in the two cases, and a non-negligible reliance on the total volume in the well. | ||
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| - | [[Image: | + | [[Image:Unipv_fu_dilution_LB.jpg| 500px|Fluorescence and dilutions LB|thumb|center|Fluorescence and dilutions in LB]] |
| - | [[Image: | + | [[Image:Unipv_fu_dilution_M9.jpg| 500px|Fluorescence and dilutions M9|thumb|center|Fluorescence and dilutions in M9]] |
| - | [[Image: | + | [[Image:Unipv_fu_dilution_LBblack.jpg| 500px|Fluorescence and dilutions LB|thumb|center|Fluorescence and dilutions in LB, black plate]] |
Revision as of 13:49, 8 October 2009
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Measurements - Fluorescence |
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Fluorescence and water dispensationDifferently from the measures of absorbance, we find out that the dispensation of water in the well causes an amplification of the fluorescence signal, both for bacterial coltures and simple liquid growing medium (in figure the results for a colture producing GFP). This amplification seams to be non linear and characterized by a kind of saturation, if analised on a large scale of volumes. You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.9, 07/07/09.
Fluorescence and dilutions in waterThe proportionality between fluorescence measurements (GFP and RFP) and different dilutions of LB was verified in two volumetric dilutions. This considerations has not to be read together with the results of the non-liner relationship between fluorescence and water dispensation, because even if different amounts of water in the well means different dilution of the colture, in that simulation we didn't work with costant volume in the well, as in this case we do. The first two measurements must not be taken into account because of the high measure's rumour. You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.18, 06/08/09.
File:Unipv fu dilution LB.png Fluorescence (GFP) and water dilution, LB
Fluorescence and volumeThe relationship between fluorescence (both GFP and RFP) and volume of LB in the well present a non-linear characteristic. This test will be done also using bacterial coltures, because we note that the behaviour of the signal from fluorescent proteins is different from that coming from the autofluorescence. You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.14, 30/07/09.
 Fluorescence and dilutions in liquid growing mediumThis experiment was done in two different growing mediums, LB and M9, and in three different total volumes in the well, 100μl, 200μl and 300μl; a black plate was also used to repeat the experiment in LB. The hypothesis of proportionality between fluorescence measurement and colture's dilutions was not verified. We observed a non-linear relationship between bacterial quantity in the well and fluorescence (both GFP and RFP) measures, in the two cases, and a non-negligible reliance on the total volume in the well. With total volumes larger than 200μl the function shows a kind of saturation, suggesting not to work with those volumes. The situation gets a little bit better using the black-home-made plate. You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.20, 13/08/09, Test n.20 "M9", 28/08/09 and Test n.20 "black", 25/08/09.
File:Unipv fu dilution LB.jpg Fluorescence and dilutions in LB File:Unipv fu dilution M9.jpg Fluorescence and dilutions in M9 File:Unipv fu dilution LBblack.jpg Fluorescence and dilutions in LB, black plate
Temporal evolution of fluorescence measurementsWe observed a particular descending evolution in fluorescence measurements, with regard to the signal of the growing medium. This behaviour depends in part on temperature, as it changes its evolution with temperature's changes (see the figure below), and probably in part on evaporation of the volume in the well. You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.14, 30/07/09.  Bleaching in fluorescent colturesA simple experiment was made to understand if the temporal descending evolution of fluorescence measurement is related to the bleaching phenomenon of the fluorescence signal or not. The results (see the figure) let us exclude this hypothesis. You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test "bleaching", 31/08/09.
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