Team:ESBS-Strasbourg/Results/Characterization
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If we correlate the fluorescence with the optical density, we can obtain a difference fluorescence factor of: 15,47. It means that the PIF6-linker-GFP gives rise to 15,5 times more fluorescence per OD unit than the normal GFP. | If we correlate the fluorescence with the optical density, we can obtain a difference fluorescence factor of: 15,47. It means that the PIF6-linker-GFP gives rise to 15,5 times more fluorescence per OD unit than the normal GFP. | ||
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Chart 1 : Relative specific fluorescence of GFP-, GFP and PIF6-linker-GFP expressing strains. | Chart 1 : Relative specific fluorescence of GFP-, GFP and PIF6-linker-GFP expressing strains. | ||
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Figure 2 : Pictures of the SDS gel (on the left), and the corresponding immunoblot <br>(on the right). The immunoblot uses rabbit anti-GFP antibodies. | Figure 2 : Pictures of the SDS gel (on the left), and the corresponding immunoblot <br>(on the right). The immunoblot uses rabbit anti-GFP antibodies. | ||
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+ | The immunoblot clearly shows a strong quantity/expression of GFP for the PIF6-linker-GFP construct. We can see that about 8 spots are recognized by anti-GFP antibodies. However for the GFP itself, there is no specific band… Nevertheless, it still allows us to answer to the hypotheses that we made earlier. | ||
+ | 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. | ||
+ | Although this discovery was fascinating, this was not good news for us since our system is based on the ability of PIF6 to bind to Phytochrome B. Truncated PIF6 would thus lead to an inefficient tagging system. | ||
+ | Nevertheless, this could be solved by fusing PIF6 sequence downstream GFP instead of upstream. | ||
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Revision as of 00:59, 28 October 2010
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