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.
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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.
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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.

Revision as of 01:27, 28 October 2010

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ESBS - Strasbourg



  

  
Let me guide you
PIF6-linker-GFP

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 fusion protein had a increased fluorescence efficiency 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.




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.


Chart 1 : Relative specific fluorescence of GFP-, GFP and PIF6-linker-GFP expressing strains.


Simultaneously, we wanted to quantify the amount of overexpressed protein (either GFP or PIF6-linker-GFP) with a SDS-PAGE (see figure 2).



Figure 2 : Pictures of the SDS gel (on the left), and the corresponding immunoblot
(on the right). The immunoblot uses rabbit anti-GFP antibodies.


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.

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. 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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