Team:Paris Bettencourt/GFP diff

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<h1>GFP diffusion</h1>
<h1>GFP diffusion</h1>
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<h2>The original experiment</h2>
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<h2>Design of the experiment</h2>
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<p>The keystone experiment of the Dubey and Ben-Yehuda paper <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">[1]</a> is a simple experiment. We use one strain of <i>gfp + B.subtilis</i> and one <i>gfp - B.subtilis</i> strain. The two strains of <i>B.subtilis</i> are put together (ratio 1:1) on an LB-agarose (1.5%) plate in exponantial phase. The goal is to have a <em>monolayer of densely packed cells.</p>
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<p>The key experiment of the Dubey and Ben-Yehuda paper <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff#references">[1]</a> is simple. They used one strain of <i>gfp + B.subtilis</i> and one <i>gfp - B.subtilis</i> strain. The two strains of <i>B.subtilis</i> in exponential phase were mixed (ratio 1:1) on an LB-agarose (1.5%) slab and sealed in a microscope slide cavity. The aim is to obtain a <em>monolayer of densely packed cells</em>.</p>
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<p>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 <i>gfp +</i> cells towards the <i>gfp-</i> cells. This cell-to-cell communication was previously unheard of and the original paper <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">[1]</a> strongly suggest that the so-called nanotubes observed through electronic microscopy by the authors is the reason of this transfer.</p>
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<center><img src="https://static.igem.org/mediawiki/2011/b/b4/Gfp_diff.png" style="width:800px"></center>
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<h2>Our experiment</h2>
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<center>GFP diffusion through the nanotubes</center>
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<p>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.</p>
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<p>The plated bacteria were then observed by time-lapse fluorescent microscopy. After a while (between 15 minutes and 2 hours), a transfer of GFP can be observed from the <i>gfp +</i> cells towards the <i>gfp-</i> cells. This cell-to-cell communication was previously unheard of and the original paper <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">[1]</a> strongly suggest that the nanotubes observed through electronic microscopy by the authors are the reason of this transfer.</p>
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<p>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.</p>
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<h2>Our results</h2>
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<p>Finally, after several tries, we managed to <em>reproduce the result expected: GFP diffusion between cells!</em></p>
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<p>We began our experiments with PY79 <i>B.subtilis</i> strain (as in the original paper) but observed that the fluorescence  of this strain was weak. We then chose to work with a 3610 strain which showed stronger fluorescence .</p>
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<p>We <em>reproduced the result expected: GFP diffusion between cells!</em></p>
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<center><img src="https://static.igem.org/mediawiki/2011/1/1e/Fluofordummies.png" style="width:800px"></center>
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<br>
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<p>You can find out more about this exciting day in the <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/09/15/">Notebook</a>.</p>
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<p>Regarding the microscopy protocol you can refer to this <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/Microscopy">page</a>.</p>
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<p>You can find the video of our results on this <a href="http://www.youtube.com/watch?v=OwURHcJYPBo">link</a>.</p>
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<center><iframe width="420" height="315" src="http://www.youtube.com/embed/OwURHcJYPBo" frameborder="0" allowfullscreen></iframe></center>
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<p>The important time steps of this experiment can be found below:</p>
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{| border="1" class="wikitable" style="text-align: center;"
{| border="1" class="wikitable" style="text-align: center;"
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|+3610 gfp-/3610 gfp+ in exponantial phase at 37°C
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|+3610 gfp-/3610 gfp+ in exponential phase at 37°C
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|-
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|[[File:0914_3610_classic_w1TRANS_s5_t1.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=0min position 5 (trans image)]]
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|[[File:0914_3610_classic_w1TRANS_s5_t1.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=0 min position 5 (trans image)]]
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|[[File:0914_3610_classic_w2GFP_s5_t1.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=0min position 5 (gfp image)]]
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|[[File:gfp_diff_fluo_0001.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=0 min position 5 (gfp image)]]
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|-
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|[[File:0914_3610_classic_w1TRANS_s5_t9.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=45min position 5 (trans image)]]
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|[[File:0914_3610_classic_w1TRANS_s5_t9.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=45 min position 5 (trans image)]]
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|[[File:0914_3610_classic_w2GFP_s5_t9.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=45min position 5 (gfp image)]]
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|[[File:gfp_diff_fluo_0002.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=45 min position 5 (gfp image)]]
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|-
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|[[File:0914_3610_classic_w1TRANS_s5_t12.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=60min position 5 (trans image)]]
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|[[File:0914_3610_classic_w1TRANS_s5_t12.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=60 min position 5 (trans image)]]
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|[[File:0914_3610_classic_w2GFP_s5_t12.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ at 37°C t=60min position 5 (gfp image)]]
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|[[File:gfp_diff_fluo_0003.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ at 37°C t=60 min position 5 (gfp image)]]
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|-
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|[[File:0914_3610_classic_w1TRANS_s5_t23.png|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=180min position 5 (trans image)]]
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|[[File:0914_3610_classic_w1TRANS_s5_t23.png|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=180 min position 5 (trans image)]]
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|[[File:0914_3610_classic_w2GFP_s5_t23.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=180min position 5 (gfp image)]]
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|[[File:gfp_diff_fluo_0004.jpg|450px|thumb|center|3610 gfp- and 3610 gfp+ strains at 37°C t=180 min position 5 (gfp image)]]
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<p>You can find out more about this exciting day on the <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/09/15/">Notebook</a>.</p>
 
<h2>Conclusions of our experiment</h2>
<h2>Conclusions of our experiment</h2>
<ul>
<ul>
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<li>We have <em>evidences of a cell-to-cell GFP transfer</em>. This is probably a non-specific transfer (as GFP is not a natural protein for <i>B.subtilis</i>) and according to the Dubey and Ben-Yehuda paper <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">[1]</a>, we can strongly syspect that it is done through nanotubes.</li>
+
<li>We have <em>evidence of a cell-to-cell GFP transfer</em>. This is probably a non-specific transfer (as GFP is not a natural protein for <i>B.subtilis</i>) and according to the Dubey and Ben-Yehuda paper <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">[1]</a>, we can strongly suspect that the GFP transfer is through nanotubes.</li>
<li>Reproducing the original experiment is difficult. We were able to observe the expected behaviour in <em>very few cells</em>. This means either that the transfer is very rare or that we need to improve the preparation of the slide.</li>
<li>Reproducing the original experiment is difficult. We were able to observe the expected behaviour in <em>very few cells</em>. This means either that the transfer is very rare or that we need to improve the preparation of the slide.</li>
<li>We used a different strain (3610 instead of PY79). This means the <em>transfer is not observed only in PY79</em>.</li>
<li>We used a different strain (3610 instead of PY79). This means the <em>transfer is not observed only in PY79</em>.</li>
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<p id="references">References</p>
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<li><i>Intercellular Nanotubes Mediate Bacterial Communication</i>, Dubey and Ben-Yehuda, Cell, available <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">here</a></li>
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<div id="scroll_right"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/Microscopy"><img src="https://static.igem.org/mediawiki/2011/e/e0/Arrow-right-big.png" style="width:100%;"></a><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/Microscopy">Microscopy protocol</a></div>
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Latest revision as of 19:35, 18 October 2011

Team IGEM Paris 2011

GFP diffusion

Design of the experiment

The key experiment of the Dubey and Ben-Yehuda paper [1] is simple. They used one strain of gfp + B.subtilis and one gfp - B.subtilis strain. The two strains of B.subtilis in exponential phase were mixed (ratio 1:1) on an LB-agarose (1.5%) slab and sealed in a microscope slide cavity. The aim is to obtain a monolayer of densely packed cells.

GFP diffusion through the nanotubes

The plated bacteria were then observed by time-lapse fluorescent microscopy. After a while (between 15 minutes 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 nanotubes observed through electronic microscopy by the authors are the reason of this transfer.

Our results

We began our experiments with PY79 B.subtilis strain (as in the original paper) but observed that the fluorescence of this strain was weak. We then chose to work with a 3610 strain which showed stronger fluorescence .

We reproduced the result expected: GFP diffusion between cells!


You can find out more about this exciting day in the Notebook.

Regarding the microscopy protocol you can refer to this page.

You can find the video of our results on this link.

The important time steps of this experiment can be found below:

3610 gfp-/3610 gfp+ in exponential phase at 37°C
3610 gfp- and 3610 gfp+ strains at 37°C t=0 min position 5 (trans image)
3610 gfp- and 3610 gfp+ strains at 37°C t=0 min position 5 (gfp image)
3610 gfp- and 3610 gfp+ strains at 37°C t=45 min position 5 (trans image)
3610 gfp- and 3610 gfp+ strains at 37°C t=45 min position 5 (gfp image)
3610 gfp- and 3610 gfp+ strains at 37°C t=60 min position 5 (trans image)
3610 gfp- and 3610 gfp+ at 37°C t=60 min position 5 (gfp image)
3610 gfp- and 3610 gfp+ strains at 37°C t=180 min position 5 (trans image)
3610 gfp- and 3610 gfp+ strains at 37°C t=180 min position 5 (gfp image)

Conclusions of our experiment

  • We have evidence of a cell-to-cell GFP transfer. This is probably a non-specific transfer (as GFP is not a natural protein for B.subtilis) and according to the Dubey and Ben-Yehuda paper [1], we can strongly suspect that the GFP transfer is through nanotubes.
  • Reproducing the original experiment is difficult. We were able to observe the expected behaviour in very few cells. This means either that the transfer is very rare or that we need to improve the preparation of the slide.
  • We used a different strain (3610 instead of PY79). This means the transfer is not observed only in PY79.
  • We proved that we will be able to see the results of our designs once we test them.
Microscopy protocol

Retrieved from "http://2011.igem.org/Team:Paris_Bettencourt/GFP_diff"