Team:ESBS-Strasbourg/Results/Characterization

  

  
  
  
  

  

In the course of our experiments, we were led to express PIF-linker-GFP to check if this fusion protein was fluorescent before going into further characterization of our system. Accordingly to the green fluorescence of the strain, we successfully expressed PIF6-linker-GFP under constitutive (promotor J23100 and RBS B0034) control (PIF3-linker-GFP is still in progress). This means this upstream tagging sequence does not prevent translation of the downstream protein.
One really surprising result yet was that colonies expressing PIF6-linker-GFP were from far more fluorescent than those expressing GFP only. This came to a surprise since the expression of both proteins are controlled by the same promoter/RBS and that GFP-fusion proteins are more likely to be less fluorescent than GFP alone!


Figure 1 : Pellets of the different strains. On the left is the negative
control which expresses no GFP. In the middle is the positive control
consisting of E. coli expressing the GFP. On the right is the culture of
E. coli expressing PIF6-linker-GFP.

This led us to make two hypotheses:

-Either the fluorescence efficiency fusion protein had a better quantum yield due to a better folding/environment around its fluorophore.

-Or, most probably, PIF6-linker-GFP expression is stronger than GFP expression, due to the PIF6-linker sequence in Nter of GFP.


In order to find an answer to this question, we compared normalized fluorescence and normalized amount of fluorescent protein in each of these strains.

First we measured fluorescence of the three different lysats (GFP negative strain, GFP expressing strain and PIF6-linker-GFP expressing strain). Cells were resuspended and sonicated and the lysate was analyzed with a fluorimetre (absorption wavelenght=490nm, emission wavelenght=510nm). The test was done 3 times to give an average measurement. The table below shows the raw results.We then calculated the specific fluorescence according to the OD600, the results are also shown in the table.



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. Simultaneously, we quantified the amount of overexpressed protein (either GFP or PIF6-linker-GFP) with a SDS-PAGE (see figure 2) 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. Result of this immunoblot is also reported in the figure 2.

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