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Team:Paris Bettencourt/GFP diff
From 2011.igem.org
GFP diffusion
The original experiment
The keystone experiment of the Dubey and Ben-Yehuda paper [1] is a simple experiment. We use one strain of gfp + B.subtilis and one gfp - B.subtilis strain. The two strains of B.subtilis are put together (ratio 1:1) on an LB-agarose (1.5%) plate in exponantial phase. The goal is to have a monolayer of densely packed cells, which is not that easy.
The plated bacteria are then observed through fluorescent microscopy. After a while (between 15min and 2 hours), a transfer of GFP can be observed from the gfp + cells towards the gfp- cells. This cell-to-cell communication was previously unheard of and the original paper [1] strongly suggest that the so-called nanotubes observed through electronic microscopy by the authors is the reason of this transfer.
Our experiment
We tried for a long time to re-do this apparently simple experiment. We failed for a long time, mainly because of microscopy issues. The advice the Ben-Yehuda team kindly gave us was to concentrate the cells as much as possible but to focus on the creation of the monolayer. Plating the cells properly is actually more important than the actual concentration of the liquid mix.
We began our experiments with a PY79 strain (as in the paper) but found quickly that our fluorescent version of this strain was rather weak. We prefered to work on a 3610 strain at the end of August and the beginning of September as we has a strong fluorescence from this one.
Finally, after several tries, we managed to reproduce the result expected: GFP diffusion between cells!
