Team:EPF-Lausanne/Our Project/T7 promoter variants/selection

From 2011.igem.org

(Difference between revisions)
(Experimental Setup)
(Results)
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[[File:rgfp_lysis_comparison.png|600px|center]]
[[File:rgfp_lysis_comparison.png|600px|center]]
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The fluorescence data shows that, in both cases, the non-lysing cells kept their plasmid DNA, meaning that there was no general, unselective lysis going on. The relevant fluorescence grew over time, as expected for non-lysing cells. The negative-control culture containing RFP seems to have grown less radically than its GFP counterpart, but the difference with respect to the lysis culture is substantial (see the lower of the two graphs).  
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The fluorescence data shows that, in both cases, the non-lysing cells kept producing fluorescent protein, meaning that there was no general, unselective lysis going on. The relevant fluorescence grew over time, as expected for non-lysing cells. The negative-control culture containing RFP seems to have grown less radically than its GFP counterpart, but the difference with respect to the lysis culture is substantial (see the lower of the two graphs).  
[[File:qPCR_dnarecovery.png|600px|center]]
[[File:qPCR_dnarecovery.png|600px|center]]
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The qPCR data in this graph indicates that the content of the supernatant is indeed the expected content, with respect to the twin cultures of each flask. Where lysed cells are expected to release GFP, we find GFP in the supernatant in much higher concentrations than RFP and vice-versa in the other flask. We can thereby confirm that the right DNA is selected in each flask and finds its way into the supernatant.  
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The qPCR data in this graph indicates that the content of the supernatant is indeed the expected content, with respect to the twin cultures of each flask. Where lysed cells are expected to release GFP plasmids, we find GFP genes in the supernatant in much higher concentrations than RFP and vice-versa in the other flask. We can thereby confirm that the right DNA is selected in each flask and finds its way into the supernatant.  
[[File:CFU.png|600px|center]]
[[File:CFU.png|600px|center]]
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The colony fluorescence count, which is our other method for quantifying DNA concentration in the supernatant, also confirms  the qPCR data from the previous graph. Indeed, the ratio of RFP colonies to GFP colonies per plate in the case of cells that lyse and release RFP plasmids grows over the course of the experiment. In practical terms, the likelihood of recovering DNA that is not the desired DNA (i.e. natural cell death leading to plasmids in the supernatant) is very low.  
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The colony fluorescence count, which is our other method for quantifying DNA concentration in the supernatant, also confirms  the qPCR data from the previous graph. Indeed, the ratio of RFP colonies to GFP colonies per plate in the case of cells that lyse and release RFP plasmids grows over the course of the experiment. In practical terms, the likelihood of recovering DNA that is not the desired DNA (i.e. natural cell death leading to plasmids in the supernatant) is low.  
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Revision as of 17:52, 21 September 2011