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Team:Paris Bettencourt/Experiments/YFP TetR diffusion experiments
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<h1>Testing nanotubes with the YFP concentrator system</h1> | <h1>Testing nanotubes with the YFP concentrator system</h1> | ||
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<h2>Summary</h2> | <h2>Summary</h2> | ||
| - | + | <p>All our experiments followed our <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/Microscopy">microscopy protocol</a> when not specified otherwise.</p> | |
<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>Results for the YFP concentrator: | <div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>Results for the YFP concentrator: | ||
<ul> | <ul> | ||
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<li>We've done <i>E.coli</i> to <i>B.subtilis</i> diffusion experiments (with negative results)</li> | <li>We've done <i>E.coli</i> to <i>B.subtilis</i> diffusion experiments (with negative results)</li> | ||
| + | <li>We've done <i>B.subtilis</i> to <i>B.subtilis</i> diffusion experiments (with negative results)</li> | ||
</ul></p></b></div> | </ul></p></b></div> | ||
<br/> | <br/> | ||
| - | <h2> | + | <h2>Design overview</h2> |
| + | <center><img src="https://static.igem.org/mediawiki/2011/9/93/YFP_concentration_principle.jpg"></center> | ||
| + | <center><p>YFP:TetR/TetO array system</p></center> | ||
| + | <p>More information on the design <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion ">here</a>.</p> | ||
| + | <h2>Emitter & receiver constructs in <i>B.subtilis</i> (receiver in plasmid)</h2> | ||
| + | <p>We succeeded to integrate the TetO Array in <i>B. subtilis</i> with the multihost episomal pHM3 plasmid. <br> | ||
| + | Experiments of <i>B.subtilis</i> (chromosomal) to <i>B. subtilis</i> (TetO Array, pHM3) to follow an eventual fluorescence diffusion by nanotube and appearance of bright spots (TetO array) has been tried.</p> | ||
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| + | </html><center> | ||
| + | {| border="1" class="wikitable" style="text-align: center;" align="center" | ||
| + | |+At the beginning of the movie : t = 0 min | ||
| + | |- | ||
| + | |[[File:28_10_TetR_TetO_subti_subti6_w1TRANS_s1_t1.jpg|350px|thumb|center|Mix of <i>B.subtilis</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - Trans image]] | ||
| + | |[[File:28_10_TetR_TetO_subti_subti6_w2YFP_s1_t1.jpg|350px|thumb|center|Mix of <i>B.subtilis</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - YFP image]] | ||
| + | |} | ||
| + | {| border="1" class="wikitable" style="text-align: center;" | ||
| + | |+At the end of the movie : t = 75 min | ||
| + | |- | ||
| + | |[[File:28_10_TetR_TetO_subti_subti6_w1TRANS_s1_t10.jpg|350px|thumb|center|Mix of <i>B.subtilis</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - Trans image]] | ||
| + | |[[File:28_10_TetR_TetO_subti_subti6_w2YFP_s1_t10.jpg|350px|thumb|center|Mix of <i>B.subtilis</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - YFP image]] | ||
| + | |} | ||
| + | </center><html> | ||
<h2>Emitter construct in <i>E.coli</i> - Receiver construct in <i>B.subtilis</i> (plasmid)</h2> | <h2>Emitter construct in <i>E.coli</i> - Receiver construct in <i>B.subtilis</i> (plasmid)</h2> | ||
| - | <p>We | + | <p>We succeeded to integrate the TetO Array in <i>B. subtilis</i> with the multihost episomal pHM3 plasmid. <br> |
| - | Experiments of E. coli (pFX234 plasmid, D. Lane strains) to B. subtilis (TetO Array, pHM3) to follow an eventual fluorescence diffusion by nanotube has been tried.</p> | + | Experiments of <i>E. coli</i> (pFX234 plasmid, D. Lane strains) to <i>B. subtilis</i> (TetO Array, pHM3) to follow an eventual fluorescence diffusion by nanotube has been tried.</p> |
</html><center> | </html><center> | ||
{| border="1" class="wikitable" style="text-align: center;" align="center" | {| border="1" class="wikitable" style="text-align: center;" align="center" | ||
| - | |+At the beginning of the movie | + | |+At the beginning of the movie : t = 0 min |
|- | |- | ||
| - | |[[File:TetRTetO0_YFPTrans01.jpg|350px|thumb|center]] | + | |[[File:TetRTetO0_YFPTrans01.jpg|350px|thumb|center|Mix of <i>E. coli</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - Trans image]] |
| - | |[[File:TetRTetO0_YFPFluo01.jpg.jpg|350px|thumb|center]] | + | |[[File:TetRTetO0_YFPFluo01.jpg.jpg|350px|thumb|center|Mix of <i>E. coli</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - Fluo image]] |
|} | |} | ||
{| border="1" class="wikitable" style="text-align: center;" | {| border="1" class="wikitable" style="text-align: center;" | ||
| - | |+At the end of the movie | + | |+At the end of the movie : t = 125 min |
|- | |- | ||
| - | |[[File:TetRTetO0_YFPTrans02.jpg|350px|thumb|center]] | + | |[[File:TetRTetO0_YFPTrans02.jpg|350px|thumb|center|Mix of <i>E. coli</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - Trans image]] |
| - | |[[File:TetRTetO0_YFPFluo02.jpg|350px|thumb|center]] | + | |[[File:TetRTetO0_YFPFluo02.jpg|350px|thumb|center|Mix of <i>E. coli</i> (YFP:tetR) and <i>B. subtilis</i> (TetO Array) - Fluo image]] |
|} | |} | ||
</center><html> | </center><html> | ||
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<center><iframe width="640" height="480" src="http://www.youtube.com/embed/BTWQVm_uwcQ?rel=0&hd=1" frameborder="0" allowfullscreen></iframe><br>Movie : Experiment of mixed YFP:tetR (E. coli) & TetO Array (B. subtilis) </center> | <center><iframe width="640" height="480" src="http://www.youtube.com/embed/BTWQVm_uwcQ?rel=0&hd=1" frameborder="0" allowfullscreen></iframe><br>Movie : Experiment of mixed YFP:tetR (E. coli) & TetO Array (B. subtilis) </center> | ||
| - | <p>We cannot | + | <p>We cannot detect any tetR-YFP foci in the receiver cells specific of YFP concentration on the TetO Array. This suggests that the YFP-tetR fusion protein from <i>E. coli</i> cannot diffuse into <i>B. subtilis</i> through the potential nanotubes.<br> |
</p> | </p> | ||
<h2>Conclusions</h2> | <h2>Conclusions</h2> | ||
| - | + | <p>We investigated the presence of nanotubes by exposing our receiver TetO array in <i>B.subtilis</i> to the YFP:TetR fusion protein emitter, both in <i>E.coli</i> and <i>B.subtilis</i>. In both case, there was no appearance of bright fluorescence spots as expected with our characterization of the TetO array. So like in <em>T7 RNA polymerase diffusion experiments</em> we have no evidence of nanotube existence yet. We are currently exploring different culture conditions that could possibly trigger nanotubes formation. We are also testing different analytical methods.</p> | |
Latest revision as of 03:41, 29 October 2011
Testing nanotubes with the YFP concentrator system
Summary
All our experiments followed our microscopy protocol when not specified otherwise.
Results for the YFP concentrator:
- We've done E.coli to B.subtilis diffusion experiments (with negative results)
- We've done B.subtilis to B.subtilis diffusion experiments (with negative results)
Design overview
YFP:TetR/TetO array system
More information on the design here.
Emitter & receiver constructs in B.subtilis (receiver in plasmid)
We succeeded to integrate the TetO Array in B. subtilis with the multihost episomal pHM3 plasmid.
Experiments of B.subtilis (chromosomal) to B. subtilis (TetO Array, pHM3) to follow an eventual fluorescence diffusion by nanotube and appearance of bright spots (TetO array) has been tried.
 |  |
 |  |
Emitter construct in E.coli - Receiver construct in B.subtilis (plasmid)
We succeeded to integrate the TetO Array in B. subtilis with the multihost episomal pHM3 plasmid.
Experiments of E. coli (pFX234 plasmid, D. Lane strains) to B. subtilis (TetO Array, pHM3) to follow an eventual fluorescence diffusion by nanotube has been tried.
 |  |
 |  |
Movie : Experiment of mixed YFP:tetR (E. coli) & TetO Array (B. subtilis)
We cannot detect any tetR-YFP foci in the receiver cells specific of YFP concentration on the TetO Array. This suggests that the YFP-tetR fusion protein from E. coli cannot diffuse into B. subtilis through the potential nanotubes.
Conclusions
We investigated the presence of nanotubes by exposing our receiver TetO array in B.subtilis to the YFP:TetR fusion protein emitter, both in E.coli and B.subtilis. In both case, there was no appearance of bright fluorescence spots as expected with our characterization of the TetO array. So like in T7 RNA polymerase diffusion experiments we have no evidence of nanotube existence yet. We are currently exploring different culture conditions that could possibly trigger nanotubes formation. We are also testing different analytical methods.
