Team:ESBS-Strasbourg/Results/Characterization
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By normalizing to 1 the GFP strain fluorescence/OD600 ratio, the PIF6-linker-GFP strains has a ratio of 15,5 (see chart 1). That is to say PIF6-linker-GFP expressing strains are 15,5 times more fluorescent than GFP expressing ones. | By normalizing to 1 the GFP strain fluorescence/OD600 ratio, the PIF6-linker-GFP strains has a ratio of 15,5 (see chart 1). That is to say PIF6-linker-GFP expressing strains are 15,5 times more fluorescent than GFP expressing ones. | ||
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- | The GFP strain showed a new (or stronger) spot at around 27 kDa compared with the GFP negative control, which perfectly fits GFP's molecular weight. While the expected molecular weight of PIF6-linker-GFP is around 43 kDa, the PIF6-linker-GFP strain showed | + | The GFP strain showed a new (or stronger) spot at around 27 kDa compared with the GFP negative control, which perfectly fits GFP's molecular weight. While the expected molecular weight of PIF6-linker-GFP is around 43 kDa, the PIF6-linker-GFP strain showed several new spots when compared with the negative control, ranking from 43 to 32 kDa, which could be interpreted as truncated proteins. We thus performed an immunoblot assay using anti-GFP antibodies in order to see whether these spots were all responsible for the fluorescence within the PIF6-linker-GFP strain. |
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- | The immunoblot clearly shows a strong expression of GFP containing protein for the PIF6-linker-GFP construct. We can see that about 8 spots are recognized by anti-GFP antibodies. | + | The immunoblot clearly shows a strong expression of different GFP containing protein for the PIF6-linker-GFP construct. We can see that about 8 spots are recognized by anti-GFP antibodies. The GFP expressing strain showed the expected spot for GFP. 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. |
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+ | 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 the strains are "only" 15,5 times more fluorescent. | ||
We can conclude that PIF6-linker-GFP leads to the expression 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 expression of different N-ter truncated GFP-containing proteins, whose cumulated quantity is greater than GFP in GFP expressing bacteria. | ||
- | Presence of such truncated proteins could be explained by the high number of methionine codons | + | 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. |
- | + | Fusing PIF6 sequence downstream GFP instead of upstream could prevent one to yield such truncated proteins. | |
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Revision as of 02:12, 28 October 2010
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