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 calculated specific fluorescence by measuring both fluorescence and amount of fluorescent protein. First we measured fluorescences of three different E.coli lysats from one GFP negative strain, one GFP expressing strain and one PIF6-linker-GFP expressing strain.
The table below shows the tests made on the previews tubes. 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. We then calculated the specific fluorescence according to the OD600, the results are also shown in the table.

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