Coding
melanopsin

Part:BBa_K838002:Experience

Designed by: 2012 EPFL iGEM team   Group: iGEM12_EPF-Lausanne   (2012-09-22)


Calcium influx observation

Idea

Melanopsin is supposed to, as soon as it is exposed to light, activate a pathway which causes the calcium concentration inside the cell to increase (see the main page for more information).

We propose to use a fluorescent bio-compatible dye (Oregon Green, OGB-1/AM) whose fluorescence is proportional to the calcium concentration. By observing the cells when they are and aren't exposed to light we can check if they react to light.

Experimental Setup

We transfected the CHO (strain dg44) cells 24h earlier and added the dye using [http://2012.igem.org/Team:EPF-Lausanne/Protocol/Calcium this] protocol: 

 Note:
 This protocol is specific to OGB-1/AM (Oregon Green) and may vary if you use different dyes.
 
 Prepare your samples by measuring their PCV (or estimating the cell amount according to
 the doubling rate). Dilute them with PBS in order to have between 200 and 500 cells/µl.
 Prepare at least one well that has seed cells.
 
 For these experiments, we usually use 200'000 cells.
 
 1. Add 5 µl 80% DMSO, 20% pluronic acid to 50 g tube of OGB-1/AM to dissolve dye powder.
    Mix with 45 µl of PBS for a 1 mM solution. (This is for 10 samples.)
 
 2. Add 5 µl 1 mM OGB-1/AM to 495 µl PBS (for one sample).
 
 3. Add 500 µl directly to the cell culture medium and incubate for 45 minutes at 37 degrees.
 
 4. Centrifuge cells and aspirate culture media and OGB-1/AM. Resuspend in 750 µl of PBS.
 
 5. Centrifuge cells and aspirate PBS. Resuspend in PBS. Repeat this step twice.
 
 6. Aspirate the PBS and add fresh culture medium to the cells.
 
 7. Your cells are ready to go under the scope! Put them on a slide or in an optical well
    plate quickly to minimize the number of dead cells you observe. If the pathway you use
    is photosensitive, remember to keep the cells in the dark.

At this point we encounter problem with our setup: Melanopsin reacts to blue light (a wavelength of around 480nm) the same frequency as the excitation frequency of the dye. The second limitation is our access to advanced microscopes; the microscope we had access to couldn't pulse the fluorescence exciting light. We used the following setup to get around the problem:

The microscope sits in "normal" mode until we find a patch with enough healthy cells. At this point we start recording (1.875fps) and switch to fluorescence mode (that is to say, switch off the overhead light and open the shutter of the mercury light). Record what happens for a certain duration and repeat this with transfected and non-transfected cells. The intensity is then measured using ImageJ.

The dye (OGB-1/AM) has an exponential decrease in intensity when exposed to light at his excitation frequency, so we hope to observe a difference in the "half-life" of the intensity between non-transfected and transfected cells (where the imported calcium hopefully reduces the exponential decay of fluorescence).

As the cells come from a transient transfection, we'll hopefully have somewhere between 20-50% of the cells who express melanopsin in varying degrees(it might be more, but that's quite improbable). In other words, the expected results are the following:

  • For non-transfected cells, the intensity exponentially decreases
  • For transfected cells, most cells will behave like the non-transfected cells, but part of the cells will behave differently by having more fluorescence than normal (at least a longer half-life of the fluorescence, but possibly something different and hopefully easily distinguishable).

Note: While the different cell samples were prepared in the same way, the fact that part of the transfected cells will behave like non-transfected cells will guarantee we can compensate for differing dye concentrations.

Results

The first graph shows how the fluorescence evolves over time in non-transfected cells (-phy42).

Team-EPF-Lausanne Experimental Results Seed.png

The cells' fluorescence show an exponential decrease as expected (note: the bumps in the "TopLeft" cell are due to another cell passing over out of focus, not due to the cell changing his fluorescence).

Team-EPF-Lausanne Experimental Results Melanopsin.png

The cells can be split into two populations:

  • Exponential: Cells 1, 3 and 8
  • Other: Cells 2, 4, 5, 6, 7 and 9

Note: cell 6 could be classified in either population as it clearly does have an increase after being exposed to light, but not as much as the others.

Conclusion

The experiment confirms the predicted results. Melanopsin causes an increase of calcium inside the cell.

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