Team:ESBS-Strasbourg/Results/Characterization
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This led us to make two hypotheses:<br><br> | This led us to make two hypotheses:<br><br> | ||
- | -Either the | + | -Either the fusion protein had a increased fluorescence efficiency due to a better folding/environment around its fluorophore.<br><br> |
-Or, most probably, PIF6-linker-GFP expression is stronger than GFP expression, due to the PIF6-linker sequence in Nter of GFP.<br> | -Or, most probably, PIF6-linker-GFP expression is stronger than GFP expression, due to the PIF6-linker sequence in Nter of GFP.<br> | ||
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Simultaneously, we wanted to quantify the amount of overexpressed protein (either GFP or PIF6-linker-GFP) with a SDS-PAGE (see figure 2).<br> | Simultaneously, we wanted to quantify the amount of overexpressed protein (either GFP or PIF6-linker-GFP) with a SDS-PAGE (see figure 2).<br> | ||
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- | The immunoblot clearly shows a strong | + | 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 at least 6 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. However for the GFP itself... | ||
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 in the PIF6-sequence, which would lead to several internal initiation of translation. | Presence of such truncated proteins could be explained by the high number of methionine codons in the PIF6-sequence, which would lead to several internal initiation of translation. |
Revision as of 01:14, 28 October 2010
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