Team:ESBS-Strasbourg/Results/Characterization
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The immunoblot clearly shows a strong expression of different GFP containing proteins for the PIF6-linker-GFP construct. We can see that about 8 spots are recognized by anti-GFP antibodies. The GFP expressing strain shows the expected spot for GFP (red boxes). Nervertheless this spot is surprisingly detectable in the negative control. If it was also present in the PIF6-linker-GFP expressing strain, one could have assumed this spot was aspecific. Here most probably this spot in the negative controle is a contamination. | The immunoblot clearly shows a strong expression of different GFP containing proteins for the PIF6-linker-GFP construct. We can see that about 8 spots are recognized by anti-GFP antibodies. The GFP expressing strain shows the expected spot for GFP (red boxes). Nervertheless this spot is surprisingly detectable in the negative control. If it was also present in the PIF6-linker-GFP expressing strain, one could have assumed this spot was aspecific. Here most probably this spot in the negative controle is a contamination. | ||
- | We measured the intensities of the different bands between 43 and 32 kDa in the PIF6-linker-GFP lane using imageJ, corrected them accordingly to their molecular weights, and compared the cumulated value to the corrected intensity of the GFP spot. As a result, there was more than 30 times more fluorescent (GFP containing) proteins within the PIF6-linker-GFP strain than GFP in the GFP strain, wherease | + | We measured the intensities of the different bands between 43 and 32 kDa in the PIF6-linker-GFP lane using imageJ, corrected them accordingly to their molecular weights, and compared the cumulated value to the corrected intensity of the GFP spot. As a result, there was more than 30 times more fluorescent (GFP containing) proteins within the PIF6-linker-GFP strain than GFP in the GFP strain, wherease it is "only" 15,5 times more fluorescent. |
Thus specific fluorescence is actually 2 times lower in PIF6-linker-GFP than in GFP, but this would be counterbalanced by a huge expression. | Thus specific fluorescence is actually 2 times lower in PIF6-linker-GFP than in GFP, but this would be counterbalanced by a huge expression. | ||
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We can conclude that PIF6-linker-GFP leads to the overexpression of different N-ter truncated GFP-containing proteins, whose cumulated quantity is greater than GFP in GFP expressing bacteria. | We can conclude that PIF6-linker-GFP leads to the overexpression of different N-ter truncated GFP-containing proteins, whose cumulated quantity is greater than GFP in GFP expressing bacteria. | ||
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Presence of such truncated proteins could be explained by the high number of methionine codons within the PIF6 sequence, which would lead to several internal initiation of translation. This would mean that translation initiation is a limiting factor for expression of certain proteins, a limit here possibly by-passed by several simultaneous initiation. | Presence of such truncated proteins could be explained by the high number of methionine codons within the PIF6 sequence, which would lead to several internal initiation of translation. This would mean that translation initiation is a limiting factor for expression of certain proteins, a limit here possibly by-passed by several simultaneous initiation. | ||
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More related to our project, fusing PIF6 sequence downstream GFP instead of upstream would prevent one to yield such truncated proteins. | More related to our project, fusing PIF6 sequence downstream GFP instead of upstream would prevent one to yield such truncated proteins. | ||
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Revision as of 02:42, 28 October 2010
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