http://2014.igem.org/wiki/index.php?title=Special:Contributions/Truan&feed=atom&limit=50&target=Truan&year=&month=2014.igem.org - User contributions [en]2024-03-29T13:46:25ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/File:Ku-xlarge.jpgFile:Ku-xlarge.jpg2015-06-02T15:37:44Z<p>Truan: </p>
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<div></div>Truanhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-18T01:11:32Z<p>Truan: </p>
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<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
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<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
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<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 20px; padding-bottom:40px;"><br />
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<p class="texte">How did we validate the three modules and improve our new protocols? Click below to find out…</p><br />
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<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a> | <a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a></p><br />
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<div class="technology">Chemotaxis module</div><br />
<div class="thelanguage"><br />
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<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control. <br />
</p><br />
<br />
<p class="title2">1. Petri dishes Test</p><br />
<p class="texte"><br />
The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was adapted from <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center><br />
<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) A pipet was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.<br />
</p><br />
<br />
<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A). As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).<br />
</p><br />
<br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center><br />
<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.<br />
</p><br />
<br />
<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.<br />
</p><br />
<br />
<p class="title2">2. Plug in Pond system<br />
</p><br />
<br />
<p class="texte"><br />
This protocol was adapted from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10 mL of the culture was mixed with 15 mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center><br />
<p class="legend">Figure 3: Plug-in-pond test design.</p><br />
<br />
<p class="texte"><br />
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4). The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/f/fd/FigureFloriefig4.png" alt="Figure 4" style="width:400px"></center><br />
<p class="legend">Figure 4: Plates after 12h at room temperature.</p><br />
<br />
<p class="texte"><br />
After one hour, no tangible results were obtained. After 12h we observed halos around the 1 M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15 µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.<br />
</p><br />
<br />
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center><br />
<p class="legend">Figure 5: Chemotaxis test with WT <i>B. subtilis</i>. The upper well contains attractive compound and the lower well contains medium without any attractive compound.<br />
</p><br />
<br />
<p class="texte"><br />
Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250 mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25 mM with tetracycline and for a concentration of 250 mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.<br />
</p><br />
<br />
<br />
<p class="texte"><br />
<b>References:</b></br><br />
[1]: Claudine Baraquet. <b>Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i></b>. 2008, Université de la Méditerranée Aix-Marseille II.<br />
</p><br />
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<br />
<p class="title2">4. Capillary test between two tubes also called the tubes test</p><br />
<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a glass capillary and using an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 6: Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the welding seam that we could not stop. <br />
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 7: Scheme of the tubes system</p><br />
<br />
<p class="texte"><br />
This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.<br />
<br><br><br />
<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br><br />
- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br><br />
- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2.<br><br />
- The same process was made with xylose as a positive control.<br><br />
<br><br />
<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 8: Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we decided to test again and possibly ameliorate the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.<br />
</p><br />
<br />
<p class="title2"> 5. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol was adapted from The Imperial College 2011 iGEM team (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).<br />
<br><br />
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br><br />
- 15 µL of each chemo-attractant was pipetted.<br><br />
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br><br />
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 9: Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- When all the chemo-attractants were added, the tips were fixed on a green support. The whole process can be seen on Figure 10.<br><br />
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 10: First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria suspensions and the bacteria content within the tips was monitored with a Thoma cell under the microscope.<br><br />
<br><br />
We experienced several problems with this system:<br><br />
- The liquid level decreasing so badly during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br><br />
- The bacteria were moving, therefore preventing us from accurately counting them (but that's the point of swimming, no??? ;-)). Taking into account this problem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.<br />
<br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 11: Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 12: Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25 mM, 250 mM and 500 mM. The tested strain was <i>Bacillus subtilis</i> 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br><br />
<br><br />
Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br><br />
<br><br />
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
Hopefully, we managed to find a negative control: galactose (25 mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25 mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p><br />
<p class="texte"> The miracle arrived! We managed to prove that our WT <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p><br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been described in the litterature.</p></b><br></p><br />
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<div class="technology">Binding module</div><br />
<div class="thelanguage"><br />
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<br />
<p class="title2">1. Preliminary experiments</p><br />
<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500 µL of either Chitin Binding Buffer (CBB) or water. 100 µL of cell suspension were plated on LB medium in order to count surviving cells.</br><br />
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.<br />
</br>We observed similar survival rates between cells treated with CBB or water (these data are not shown on figure 16).<br />
Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/parts/e/e0/Data_not.jpg" width="40%"></center><br />
</br><br />
<br />
<p class="legend">Figure 16: We don't want to bother you with useless data</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br> <br />
<br />
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).<br />
There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain (Beads on Figure 17). <br />
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its binding onto chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.<br />
</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a green fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p><br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p><br />
<br />
<p class="texte">Using ImageJ software, we created 3D pictures and movies of those images.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><video width="45%" poster="https://static.igem.org/mediawiki/2014/4/4c/Video_poster.png" controls><br />
<source src="https://static.igem.org/mediawiki/2014/6/61/Beads_3D_movie.ogg" type='video/ogg; codecs="theora, vorbis"'/><br />
</video></center><br />
</br><br />
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes festival…)</p><br />
<br />
<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/9/97/Photo_lavage_microscopie.png" width="45%"></center><br />
<p class="legend">Figure 20: Microscopic view of elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right). <br />
</p><br />
<br />
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</p><br />
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<div class="technology">Fungicides module</div><br />
<div class="thelanguage"><br />
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<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5 µM, 25 µM, 100 µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.<br />
</br><br />
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100 µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper sulfate, a well-known chemical fungicide was used as a positive control. Inhibition of the fungal growth was complete with 20 mg/ml copper sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on Figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.<br />
</p><br />
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<center><img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"></center><br />
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p><br />
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</br><br />
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<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br><br />
Camille also concluded that turning the water of the Canal du Midi to deep blue using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) ! <br />
</p><br />
<br />
<p class="title2">2. Test with antifungal bacteria</p><br />
<br />
<p class="texte">In order to test our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br><br />
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate (figure 21).</br><br />
</p><br />
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</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22: Results with transformed bacteria.</p><br />
<br />
<p class="texte"><br />
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br><br />
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p><br />
<br />
<br />
<p class="title2">3. <i>In planta</i> tests with SubtiTree</p><br />
<br />
<br />
<center><img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px"/></center><br />
<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p><br />
<br />
<p class="texte"><br />
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br><br />
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br><br />
</p><br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br><br />
Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40 h. The number of necrotic sites and the lesion sizes appeared reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b><br />
</p><br />
<br />
<center><img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg"></center> <p class="legend">Figure 24: Results of <i>in planta</i> test</p><br />
<br />
<br />
<p class="texte">We now expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improve the performance of SubtiTree. Thus, these results pave the way towards the testing of SubtiTree in infected plane trees</br><br />
More than ever, let's save our trees with SubtiTree!<br />
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<p class="texte">Let's introduce you to our fabulous tree-friendly team, 100% biodegradable and certified pesticide-free.</p><br />
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<p class="title1">Students</p><br />
<p class="title3"> <i>(Move your mouse over image to enlarge!)</i> </p><br />
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<img src="https://static.igem.org/mediawiki/2014/d/da/Diane_gros.png" alt="Diane Barbay" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Diane Barbay</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Diligent (Wait a minute, I have to write down these results in my notebook!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Hardworking, Diane is the most consciencious in her work: her notebook is so clean and organized! She is persevering and always tries to find the answer to the problem (except when plasmids shortens between two digestions…). She talks to bacteria to encourage them taking up plasmid during transformation. She is like a babysitter for our cells :-)<br />
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<img src="https://static.igem.org/mediawiki/2014/0/0e/Emeline_gros.png" alt="Emeline Flajollet" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Emeline Flajollet</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in competent cells (Oh no, I have to make competent cells again…)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Emeline looks discreet at first, but when you meet her, you discover a frank person who knows how to express her opinions and how to be understood, which is a good thing for team work. She is diligent and involved in her work, always trying to do her best, and providing advice to others. She spends a lot of time on social networks, allowing us to keep in touch with other teams.<br />
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<p class="title2"><b>Mathieu Fournié</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in communication (I found someone else who wants to interview us!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Master of Microbiology at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About him:</b> Initial founder of the project, Mathieu is a fan of Midi Pyrenées's region and is eager to protect its natural environment and resources. He is devoted to the project and does his best to succeed. His leadership skills help us focus on the main goals and deadlines. Even if it makes him forget to do his own tasks! His good interpersonal skills and numerous contacts helped us to capture media attention. <br />
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<p class="title2"><b>Florie Gosseau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our IT expert (Modeling? Ok let’s do it!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Bioinformatics and Biological Systems Master at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About her:</b> Florie is a smiling and spontaneous girl. She is this kind of person capable of giving a true smile when nothing works, and she can disentangle conflicts thanks to her patience and her kindness. She is really helpful and never says no when you ask for service. She is also frank and reports problems when there are some. Finally, she is the only one good at informatics, which makes her indispensable for us! When she wears her lab coat, she is able to fly like Batman or being the queen of cats' kingdom. Really, it is wonderful to see her!<br />
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<img src="https://static.igem.org/mediawiki/2014/9/92/Camille_gros.png" alt="Camille Jourdan" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Camille Jourdan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Expert in cloning (Camille, which enzyme should I take?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About him:</b> Jovial and funny, Camille has sometimes eruptions of madness which makes him so special. Conversely, he is capable of incredible moments of intelligence and concentration (primers and plasmids hold no secret for him) and he is really diligent. He is sarcastic but definitely not nasty. He is the athlete of the team and never misses an opportunity to make gymnastics exercises or Plasmid Dance!<br />
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<p class="title2"><b>Aurélie Kanitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Distracted (Oh I forgot my keys! I have to go back home, see you in 15 minutes!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Aurélie is a joyful and spontaneous person, which makes you laugh and smile. Often distracted, she can make blunders… She is natural and honest and does not try to play a role. She is open-minded and helpful: you can count on her. Strong and sensitive at the same time, she is able to face difficulties. Don’t go by appearances, this tiny girl has personality and she does not let others walk over her!<br />
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<img src="https://static.igem.org/mediawiki/2014/2/25/Laureen_gros.png" alt="Laureen Mirassou" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Laureen Mirassou</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our best negotiator (Don’t worry about prices for flight tickets, I deal with that)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school</p> <br />
<p class="textesimple"><b>About her:</b> Laureen is a mix of kindness, helpfulness and generosity. Always trying to help people, she is essential to ease tensions and to solve social issues: she says what is wrong gently and calmly. Her good interpersonal abilities helped us to make good business and to solve many administrative problems. Her high level of English proficiency is also something precious for the team.<br />
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<img src="https://static.igem.org/mediawiki/2014/8/8d/Manon_gros.png" alt="Manon Molina" border="0" style="margin-top:5px"/><br />
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<p class="title2"><b>Manon Molina</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Organized (Let’s make a to-do-list!)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> Manon is organized: she always makes to-do-lists to not forget anything and do tasks in time. Thank goodness, she is here to monitor the budget and do the accounting! She makes her work conscientiously and meticulously, not allowing mistakes. She is kind and lenient but can also be frank when she does not endorse your methods. In short: serious and devoted to the project.<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
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<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/5/5c/Fanny_gros.png" alt="Fanny Pineau" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"><br />
<p class="title2"><b>Fanny Pineau</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Absent-minded (which cloning are we doing?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About her:</b> In the lab, Fanny is often lost in her works: too many cloning at the same time, you should not ask too much to her blond brain! Not enough concentrated, she can sometimes make blunders. Besides that, she is applied and perfectionist when she plunges into her work. Finally, she is a joyful and smiling person; also honest and frank, telling it like it is.<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div class="zoomb"><div style="float:right; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/0/0d/Pierre_gros.png" alt="Pierre Reitzer" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Pierre Reitzer</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Relaxed (Make a presentation barefoot, why not?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Biochemistry Master at INSA Toulouse engineering school </p> <br />
<p class="textesimple"><b>About him:</b> Pierre is the co-founder of the project. He is really friendly and is always willing to discuss and help people with their experiments. Moreover, he greets us with a smile each time we see him in the lab. He is spontaneous and forthright: we know what he thinks. Sometimes, he has his head in the clouds and fails the same experiment three times… But he is involved in his work and does not count hours. <br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<div class="zooma"><div style="float:left; width:215px;"><br />
<p class="title2"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Abdel_gros.png" alt="Abdel Touré" border="0" style="margin-top:5px"/><br />
</div><br />
<div style="float: right; width:720px;"><br />
<p class="title2"><b>Abdel Touré</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Luxury tastes (Why shouldn't we go to the Hilton?)</p><br />
<p class="textesimple"><b>Studies:</b> First year of Bioinformatics and Biological Systems Master at Paul Sabatier University of Toulouse</p> <br />
<p class="textesimple"><b>About him:</b> Abdel is this kind of person that you remember: he is always jovial and running everywhere. He is a funny guy who likes making jokes. But, be careful! He is also sensitive, your jokes might not make him laugh at all. He takes care of his appearance, and likes smoking his e-cigarette. He always swears in English, but we like it because this is funny, and he is a good English speaker!<br />
</p></p><br />
</div></div><br />
<br />
<div class="clear" style="margin-top:85px;"></div><br />
<br />
<p class="title1">Instructors</p><br />
<br />
<div style="float:left; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b6/Advisor_Gilles.jpg" style="margin-top:5px"/><br />
</div> <br />
<br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Gilles Truan</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Senior Researcher at CNRS (Centre National de la Recherche Scientifique) and at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
<p class="textesimple"><b>About him:</b> He is with us from the beginning to the end as a father (and already a grandfather!!), our instructor is the master of cloning and the captain of PCR! He provided us with valuable help and good tips he learned at the time he was supervising Marie Curie's doctoral thesis.</p><br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/Advisor_Brice.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Brice Enjalbert</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The right-hand man of our amazing instructor!</p><br />
<p class="textesimple"><b>Job:</b> Lecturer at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:left; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Advisor_Florence.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="title2"><b>Florence Bordes</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> The only female advisor! </p><br />
<p class="textesimple"><b>Job:</b> Lecturer at LISBP (Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés).</p> <br />
</div><br />
<br />
<div class="clear" style="margin-bottom:60px;"></div><br />
<br />
<div style="float:right; width:215px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2d/Advisor_Cam.jpg" style="margin-top:5px" /><br />
</div> <br />
<br />
<div style="float:left; width:700px; margin-left:5px; margin-right:40px"><br />
<p class="title2"><b>Kaymeuang Cam</CENTER></b></p><br />
<p class="textesimple"><b>Feature:</b> Our bacteriologist advisor! </p><br />
<p class="textesimple"><b>Job:</b> Lecturer at IPBS (Institut de Pharmacologie et de Biologie Structurale)</p> <br />
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<h2>Modeling</h2><br />
<p>To develop a predictive model</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modeling</p> <br />
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<p class="texte"><br />
<br />
Modeling is a tool used to simplify and study systems. It helps us to predict behavior of biological systems using bibliographic or experimental data.</br><br />
<br />
The following modelisation focuses on the development of a bacterium in trees. The bacterial growth in trees seems to be unknown, thus we must infer <i>Bacillus subtilis'</i> behavior.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Aim<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a tree endophyte strain. A study showed that <i>Bacillus subtilis</i> could develop and fully colonize a tree, reaching a concentration of 10<sup>5</sup> cells per gram of fresh plant. We need to know in which conditions the growth of <i>B. subtilis</i> is optimum in a tree and if the weather can stop its development during winter. Therefore we decided to work on the growthof <i>Bacillus subtilis'</i> in function of the temperature during the year. <br />
<br>Modeling bacterial growth in a tree section generates some difficulties. We need to know the distance between two tree extremities (treetops and root) or the speed sap flow. However the flow of speed sap can vary with temperature during the day. The composition of sap also varies due to seasons and type of container (phloem, xylem). Furthermore a tree is not an homogeneous system: its roots, trunk and branches do not contain the same amount of sap and wood. <br>The average speed of the plane tree sap is 2.4 m/h, which means that in a day the sap of a 30 m tree will flow from one extremity to the other. We thus reduced the tree to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji</b> (See References), after six months of <i>Bacillus subtilis</i> growth in a tree, bacteria cells reach a concentration of 10<sup>5</sup> cells per gram of fresh plant. We assume that 10<sup>5</sup> cells/g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impacts on bacterial growth.<br />
</li><br />
<li><br />
It is assumed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. The composition sap used was the one from birch sap.<br><br />
In these conditions, the growth rate µ is optimal. From this value we can extrapolate a growth curve as a function of temperature. We used the <b>cardinal temperature model</b>: </p><br />
<br />
<center style="margin-bottom:50px;"><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature</br><br />
µ<sub>opt</sub>: Optimal growth rate</br><br />
µ: growth rate at temperature T</br><br />
T<sub>max</sub>: Maximum temperature supported by bacteria</br><br />
T<sub>min</sub>: Minimum temperature supported by bacteria</br><br />
T<sub>opt</sub>: Optimum temperature for the growth</br></br><br />
<br />
Necessary parameters for this function are minimun temperature T<sub>min</sub> and maximum temperature T<sub>max</sub>, optimal temperature for the growth T<sub>opt</sub> and optimal growth rate µ<sub>opt</sub>.</br><br />
</br><br />
T<sub>min</sub>: 10°C</br><br />
T<sub>max</sub>: 52°C</br><br />
T<sub>opt</sub>: 37°C</br><br />
µ<sub>opt</sub>: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth rate (µ<sub>opt</sub>) is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C (according to growth tests performed at 10°C and 4°C under similar conditions for the measurement of µ<sub>opt</sub>), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br><br />
Conditions applied:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center style="margin-top: -52px;"><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Figure 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa</b> (See References) is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas:</p><br />
<center style="margin:-44px 0 65px;"><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our case, the growth rate µ depends on the temperature. <br />
<br>N corresponds to the bacterial population, N<sub>min</sub> and N<sub>max</sub> are two asymptotes. <br />
<br>The parameter m is a curvature parameter. Larger m is, smaller is the curvature of the deceleration phase with the model. <br />
<br>The parameter n is a parameter related to the period lag. Larger n is, shorter is the period of lag. <br />
<br><br />
<br>N<sub>min</sub> is slightly lower than N<sub>0</sub>. When N is small at the initial state (N = N<sub>0</sub>) <i>i.e.</i> N is close to N<sub>min</sub>, N<sub>min</sub>/N is almost equal to 1. Therefore the term (1-(N<sub>min</sub>/N)) is nearly 0 and the growth is very slow. <br />
<br>If N decreases until it reaches N<sub>min</sub>, the term (1-(N<sub>min</sub>/N)) is equal to 0. Therefore the growth is null.<br />
<br> Similarly when N is equal to N<sub>max</sub>, the term (1-(N/N<sub>max</sub>)) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this, we worked under two conditions: positive and negative growth. Theses conditions can be translated in two equations. This leads to the writing of this model:</p><br />
<center style="margin: 65px 0;"><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
The term (1-(Nmin/N)) is not taken into account when there is growth. <br>The term (1-(N/Nmax)) is not taken into account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region during the years 2011-2013 are used to calculate average daily temperatures. Thus we can determine <i>B. subtilis</i> growth in a tree located in Toulouse during a year. This values are obtained for each day by the average on the highest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650 kg/m³. The average diameter of the trunks of the concerned trees is about 0.80 m and they are 15 m high. This represents a volume of 30 m³. Therefore the weight of the trunk is 19.604 kg.<br />
We need to add to this weight the weight of branches, twigs, about 25% of leaves and about 15% of roots (<a href="http://www.guichetdusavoir.org/viewtopic.php?t=25895">source-FR</a>).<br />
</br><br />
<!--pas compris ces deux dernières phrases--><br />
The average weight of a tree plane is 27,446kg. We inoculated 10 mL of bacterial culture at 10<sup>9</sup>cfu/mL, <i>i.e.</i> 10<sup>10</sup> bacterial cells. This represents 3.64x10<sup>2</sup>cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Figure 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) <i>Bacillus subtilis</i> growth curve during one year (N is cell quantity by g of fresh plant). (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10°C.</p><br />
<br />
<p class="texte"><br />
In our model, growth starts only from 10°C, which happens between March and April. This period seems to be the most suitable to administer the strain in the tree. Starting in December the temperature decreases below 4°C, corresponding to the threshold below which bacteria starts to die. <br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, temperature variations are certainly lower in trees than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate and nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also induces a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change of temperature, the rise of sap in the trunk and sap composition variations. All these parameters can consequently slow-down or boost the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached (See Annexe).<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<br />
<ul><br />
<li class="tree"><p class="texte"><br />
Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) <b>Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain.</b> FEMS Microbiol Ecol 65: 565–573<br />
</li></p><br />
<li class="tree"><p class="texte"><br />
A. Garnier(1977) <b>Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques.</b> Ann. Sci. Foresi. 34 (1): 17-45<br />
</li></p><br />
<li class="tree"><p class="texte"><br />
Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) <b>Composition and properties of birch syrup (Betula pubescens).</b> J. Agric. Food Chem 37 (1): 51–54<br />
</li></p><br />
<li class="tree"><p class="texte"><br />
L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN <b>Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model.</b> J. theor. Biol. 162 : 447-463<br />
</li></p><br />
<li class="tree"><p class="texte"><br />
Hiroshi Fujikawa (2010), <b>Development of a New Logistic Model for Microbial Growth in Foods.</b> Biocontrol of Science Vol 15: 75-80<br />
</li></p><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
<p class="texte"> To download the script and the table <a href="https://static.igem.org/mediawiki/2014/0/01/Annexes.zip">Click Here</a></p><br />
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{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/Project/project-contextTeam:Toulouse/Project/project-context2014-10-17T23:18:09Z<p>Truan: </p>
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<h2>Project context</h2><br />
<p>A threatened heritage</p><br />
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<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Context project</p> <br />
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<p class="texte">One of Europe most famous scenic is without a doubt the Toulouse Canal du Midi. Situated in the heart of our city and going through Southern France, our lab is only 5 minutes walk from the Canal du Midi. Everyone in the team enjoys the quietness and loveliness of this charming place. Unfortunately, this heritage is threatened: a phytopathogenic fungus, <i>Ceratocystis platani</i>, is devastating the trees lining the Canal.<br />
</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/14/Overview_context.jpg"/></center><br />
<br></br><br />
<br />
<p class="texte">The situation is alarming: today the only solution is preventive tree cutting to stem the fungal epidemic. The consequences are disastrous. Not only for the beauty of the landscapes but it has also a huge environmental, social and economic costs. Today, 42,000 plane trees are threatened, knowing that cutting and planting a new resistant tree is about € 4,000.<br />
</p><br />
<br />
<p class="title1">The Canal du midi</p> <br />
<div style="float:right; margin-left:40px"><img style="width:300px;" src="https://static.igem.org/mediawiki/2014/0/04/Canal_automnal.jpg"></div><br />
<p class="texte" style="display:block; float: left; width: 600px;">This ingenious masterpiece respects the environment in which it is harmoniously integrated since 1681 by Pierre-Paul Riquet.<br />
Characterized as a summit level canal, it culminates at 189 meters of altitude and connects, from coast to coast the Atlantic and the Mediterranean seas.<br />
On December 7<sup>th</sup> 1996, the UNESCO registers Le Canal du Midi on the list of world’s legacy. The first plane trees bordering the canal were planted after its creation in 1776. Resolving the erosion of riverbanks and the evaporation of the water, the plane imposed itself as a dominant and emblematic tree. The wooded legacy is estimated to about 42,000 trees, it contributes to the landscape and aesthetic.<br />
Today, the canal du midi is primarily used for tourism, recreation and housing. Busier than the Seine (Paris river), it accounts for one fifth of French river tourism and 80 % tourists are foreigners. Boaters seeking for tranquility, quietness and a unique environment mainly navigate over it.<br />
</p><br />
<br />
<div class="clear"></div><br />
<br />
<p class="title1">Canker Stain</p> <br />
<div style="float:left; width:215px; margin-right:40px"><img style="width:200px; float:left;" src="https://static.igem.org/mediawiki/2014/5/5e/Ceratocystis.jpg"></div><br />
<p class="texte">The canker stain is a disease caused by <i>Ceratocystis platani</i>, a microscopic fungus that exclusively attacks Plane Trees.<br />
Probably introduced in France by infected wooden munitions cases coming from United States of America in 1945, the fungus introduces itself inside the sain tree’s heart, blocks the sap flow and kills it in only 2 to 5 years.<br />
On the regulatory side, on July 31<sup>th</sup> 2000 a ministerial order has classed <I>Ceratocystis platani</I> as an harmful organism for plants. This order leads to a mandatory fight against the fungus.<br />
</p><br />
<br />
<br></br><br />
<br />
<p class="title1">Fighting the plane trees pathogen</p> <br />
<p class="texte">Regarding this imminent threat, some solutions emerged.<br />
</br>First of all, chemical fungicides were used. However, the community realized it was impossible to eradicate the whole fungus. <br />
</br>Therefore, the French Institut National de Recherche Agronomique (INRA) created a new type of plane trees named Platanor which were resistant to the fungus infection. Unfortunately, this extreme measure could not save the already contaminated trees nearby the Canal du Midi.<br />
</br>Today, the only option remains in the tree-cutting of the contaminated plane trees. Nevertheless, this previous solution is not optimal because of the high cost and the major touristic and social impact for our heritage.<br />
</p><br />
<br />
<p class="title1">References</p><br />
<ul><br />
<br />
<li class="tree"><p class="texte">Photo VNF/Sud-Ouest</p></li><br />
</ul><br />
<br />
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{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/Team/Fun_factsTeam:Toulouse/Team/Fun facts2014-10-17T23:05:52Z<p>Truan: </p>
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<img src="https://static.igem.org/mediawiki/2014/3/30/Kilometers.jpg" style="margin-top:5px; width:470px" /><br />
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<br />
<p class="title2">Have you ever tried to estimate the kilometers traveled by your team during this summer?</p><br />
<p class="texte"><br />
According to our calculations, one team member walks approximately 4 kilometers per day all around the <br />
laboratory. This represents 5,544 kilometers for the whole team during the 126 days of this epic <br />
adventure. <br />
What does that mean exactly? Simply that we could walk, cycle, swim or whatever you want to <br />
Kazakhstan, Russia, Kenya… We could even have reached the USA but we decided to stay in the lab <br />
and take the plane to go to Boston!</p><br />
<br />
<br> </br><br />
<br />
<div style="float:right; width:500px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/52/Interview.jpg" style="margin-top:5px; margin-left: 62px; width:450px" /><br />
</div> <br />
<br />
<p class="title2">What do trees lining the “Canal du Midi” think about SubtiTree?</p><br />
<p class="texte"><br />
According to our homemade impartial survey, 94% of the questioned plane trees approve our project and are interested in <br />
serving as guinea pigs for our new bacterial medicine. This percentage represents 41,580 trees which <br />
are also gathered in the association called: “Happy tree friends“.</p><br />
<br />
<br />
<div style="float:left; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Multidisciplinary_yes_we_are.jpg" style="margin-top:5px; width:450px" /><br />
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<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br />
<p class="title2">Multidisciplinary… Yes we are!</p><br />
<p class="texte">Housework in our laboratory became necessary when most of people were in vacations except us. <br />
But do you know the novelty this year in our team? Times are changing because now men are <br />
cleaning! ;-)</p><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br> <br> <br><br />
<br />
<div style="float:right; width:500px; margin-top:50px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/World_cup.jpg" style="margin-top:5px; width:450px; margin-left:62px;" /><br />
</div> <br />
<br> <br> <br> <br> <br> <br><br />
<p class="title2">The “can’t miss it” event of the summer: the 2014 Football (also called Soccer in some third zone countries!!) World Cup!</p><br />
<p class="texte">From 06/12/14 to 07/13/14, the Toulouse iGEM Team was cheering for the French team. During the <br />
games of the French team, our group was juggling with wet lab and large screen projections! Despite <br />
our support, the French team did not win the World Cup. However, we have not said our last world <br />
yet: let’s see what will happen in 2018… ;-)</p><br />
<br />
<div class="clear"></div><br />
<br />
<div style="margin-top:50px; text-align:center;"><br />
<p class="title2">Have you ever...</p><br />
<p class="texte" style="text-align:center;">...forgotten a tube culture or a petri dish?<br />
<br>Never? Let us show you what happens in that case!</p><br />
</div><br />
<center><br />
<table><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/f/fa/Old_tube1.png" width="100px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/1/1b/Old_tube2.JPG" width="200px"></td></tr><br />
<tr><td><img src="https://static.igem.org/mediawiki/2014/d/d4/Old_petri1.png" width="270px"></td><br />
<td><img src="https://static.igem.org/mediawiki/2014/d/d7/Old_petri2.JPG" width="300px"></td></tr><br />
</table><br />
</center><br><br><br />
<p class="texte" style="text-align:center;">...tried to make a 3% agarose gel?</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/5a/3%25_gel.jpg" width="200px"></center><br />
<br><br />
<br><br />
<br> <br />
<br />
<div style="text-align:center; width:760px; margin:0 auto; margin-top:15px; border-top:1px solid #555; padding-top:60px;"><br />
<p class="title2" style="padding-bottom:15px;">To finish this part, let’s do the official Awards Ceremony of the 2014 Toulouse iGEM Team!</p><br />
<ul><br />
<li class="tree"><p class="texte">The Geek Award goes to... <b>Florie</b> <br />
who spent the longest time in front of her laptop for the modeling part!</p></li><br />
<li class="tree"><p class="texte">The latest-survivor-of-weekly-meetings goes to... <b>Diane</b> who stayed up until 3am because she was skyping from South Korea!<br />
</p></li><br />
<li class="tree"><p class="texte">The worst singer award goes to ...<b>Abdel</b> who <br />
spent the whole day singing badly in the lab!</p></li><br />
<li class="tree"><p class="texte">The dancer award goes to ... <b>Camille</b><br />
who did the famous Plasmid Dance!</p></li><br />
<li class="tree"><p class="texte">The “hello you” award goes to... <b>Pierre</b> who was always saying <br />
“Hello you” each time he meets someone (approximatively 256 times per day)!</p></li><br />
<li class="tree"><p class="texte">The most tired award goes to...<b>Manon</b> but we still do not know why!</p></li><br />
<li class="tree"><p class="texte">The misplaced ideas award goes to... <b>Mathieu</b> but you do not want to know why!</p></li><br />
<li class="tree"><p class="texte">The perseverance award goes to... <b>Emeline</b> who succeeded a cloning after twelve trials!</p></li><br />
<li class="tree"><p class="texte">The drawing award goes to... <b>Fanny</b> <br />
who drew our first SubtiTree logo!</p></li><br />
<li class="tree"><p class="texte">The best phone-caller award goes to...<b>Laureen</b><br />
who was our perfect lab secretary!</p></li><br />
<li class="tree"><p class="texte">The biggest blunder in the lab award goes to ... <b>Aurélie</b> who poured an agarose gel without gel tray!</p></li><br />
<li class="tree"><p class="texte">And last but not least ... The Best Nervous breakdown Award goes to ... <b>Our -20°C freezer</b>! The whole team is grateful for its hard work during a hot summer! Three successive breakdowns during the hot days of summer: thank you freezer, so long chap, we’ll unplug you after iGEM is finished and you’ll retire, hopefully not in the Canal du Midi…</li></p><br />
</ul><br />
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{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/Acknowledgements/SponsorsTeam:Toulouse/Acknowledgements/Sponsors2014-10-17T23:00:51Z<p>Truan: </p>
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<p style="color: #696969; padding-top: 20px; font-size: 16px; float: left;">Acknowlegdements&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Sponsors</p><br />
<ul class="topnav" id="topnav" style="top: 15px;"><br />
</ul><br />
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<p class="texte"><br />
Financial support is mandatory for any iGEM team. All of our sponsors are committed to promoting the scientific and innovative experiences such as the iGEM project. We would like to thank our collaborators for their contribution to our work by giving us an outstanding overview and help. The iGEM adventure could not have been made without their support and their faith in our project.<br />
<br><br />
Thanks to the public and private sponsors, the Toulouse iGEM team collected <b>€ 60,082.73 </b>. The encouragement was based on financial support from our schools and other partners, gifts of consumables or special sale prices on laboratory equipment.<br />
</p><br />
<br />
<p class="title1">Our institutions</p><br />
<br />
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<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/9/97/INSA_Toulouse_new_logo.jpg"><br />
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</td><br />
<td colspan="3" width="100%"><br />
<p class="texte"><br />
INSA Toulouse (Institut National des Sciences Appliquées) is a famous engineering school allowing the specialization in eight different subjects such as civil engineering, biochemistry but also informatics. The school was created in 1953 and hires today 220 teachers and researchers and has 8 research laboratories.<br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td colspan="3" width="100%"><br />
<p class="texte">L'Université Toulouse III Paul Sabatier is a multidisciplinary University involved in sciences and technologies, sport, gestion and communication but also health and medical studies. Funded in 1229, this University is one of the oldest in Europe.</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/7/7f/Logo_UPS.jpg"></td><br />
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<tr> <td colspan="4" width="100%"><br />
<p class="title1">Our major supports</p><br />
</td></tr><br />
<tr><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/6/60/Logo_Minist%C3%A8re.gif"></td><br />
<td colspan="3"><br />
<p class="texte"><br />
Le Ministère de l'Agriculture, de l'Agroalimentaire et de la Forêt (MAAF) was created in 1881 and implements the government's policies in agriculture, food market industries and forest.<br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
Voies Navigables de France (VNF) is a French public establishment of an administrative nature created in 1991 which aims to manage the major part of the navigable waterways in France.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/e/e2/Logo_VNF.jpg"></td><br />
</tr><br />
<tr><td colspan="4" width="100%"><br />
<p class="title1">Our sponsors</p><br />
</td></tr><br />
<br />
<tr><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/5/5f/Logo_LISBP.png"></td><br />
<td colspan="3"><br />
<p class="texte"><br />
The Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP) was created in 2007 and is based on the fusion of the Laboratory of Biotechnology and Bioprocesses and the Laboratory of Engineering in Environmental Processes. The LISBP is mostly specialized in innovative technics regarding living and process sciences such as microbiology, biocatalysis and separation technics.<br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
Toulouse White Biotechnology (TWB) is a French infrastructure developing new technics of sustainable production by enhancing the use of renewable carbon source. TWB has three main missions: promoting the white biotechnologies, improving the scientific innovation, binding the fundamental research and the industrial world. White biotechnologies are for industrial goals, including the use of cells and enzymes to create industrial products.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/4/4a/Logo_TWB.png"></td><br />
</tr><br />
<br />
<tr><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/0/05/Logo_Adisseo.jpg"></td><br />
<td colspan="3"><br />
<p class="texte"><br />
Adisseo is a subsidiary of Bluestar Company and a worldwide expert in animal nutrition. This branch has a major role in the improvement of food chain while preserving animals, mankind and environment.<br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
The Genopole of Toulouse (GenoToul) was created in 1999 to develop genomes studies, bioinformatics and to promote the innovation by encouraging interdisciplinary projects. GenoToul welcomes around fifty projects each year regarding many subjects such as healthcare, agronomy, biotechnologies, ecology and environment.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 180px;" src="https://static.igem.org/mediawiki/2014/6/61/Logo_Genotoul.png"></td><br />
</tr><br />
<br />
<tr><br />
<td width="180px"><br />
<img src="https://static.igem.org/mediawiki/2014/0/0d/Logo_COMUE.jpeg"><br />
<td colspan="3"><br />
<p class="texte"><br />
The Communauté d'Universités et d'Etablissement de Toulouse (COMUE) includes 14 establishments such as universities, engineering schools, specialised schools. It aims to position the Toulouse University at the best european and international level.<br />
</p><br />
</td><br />
</tr><br />
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<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
Labex Tulip is a laboratory composed of five units specialized in agrobiosciences and environment. It represents an enormous research potential with approximately 400 people belonging to many different laboratories. Labex Tulip is focused on the interactions between scientific institutions and the scientific community. The main goal is to enhance the research studies and the attractivity of its laboratories.<br />
</p><br />
</td><br />
<td width="180px" ><br />
<img style="width: 90px;" src="https://static.igem.org/mediawiki/2014/5/5d/Logo_LabexTulip.jpg"><br />
</tr><br />
<br />
<tr><br />
<td width="180px"><br />
<img style="width: 100px;" src="https://static.igem.org/mediawiki/2014/5/54/Logo_CROUS.jpg"></td><br />
<td colspan="3"><br />
<p class="texte"><br />
The Centre Régional des Oeuvres Universitaires et Scolaires (CROUS) is a public facility controlled by the Ministery of Higher Education and Research. The main goal is to promote the good living conditions of students. The CROUS can provide help in many different actions: direct help through financial fundings, undirect help with housing and catering. <br />
</p><br />
</td><br />
</tr><br />
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<td colspan="3"><br />
<p class="texte"><br />
The Hérault Département is in the Languedoc-Roussillon region (South of France) with more than 1,000,000 inhabitants.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 80px; " src="https://static.igem.org/mediawiki/2014/8/88/Herault.jpg"><br></br><br />
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<td width="180px"><br />
<img style="width: 100px;" src="https://static.igem.org/mediawiki/2014/0/02/Castelnaudary.jpg"><br />
</td><br />
<td colspan="3"><br />
<p class="texte"><br />
Castelnaudary is a 12,000 inhabitant’s city located in Aude department (South of France) where Pierre-Paul Riquet constructed a pond named Le Grand Bassin.<br />
</p><br />
</td><br />
</tr><br />
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<p class="texte"><br />
Labastide-d’Anjou is a 1,500 inhabitants’ village located in the Aude department in the Languedoc-Roussillon region (South of France). This fortified town is situated nearby the Canal du Midi.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 70px; " src="https://static.igem.org/mediawiki/2014/4/48/Labastide_d%27Anjou.png"><br />
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</td><br />
<td colspan="3"><br />
<p class="texte"><br />
Montréal is a 2,000 inhabitants’ village located next to the Montagne noire, in the Aude department in the Languedoc-Roussillon region (South of France).<br />
</p><br />
</td><br />
</tr><br />
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<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
Sallèles d’Aude is a 3,000 inhabitants' village situated in Aude department in the Languedoc-Roussillon region (South of France) where the “Canal de jonction” runs through, joining the Canal du Midi and Canal de la Robine.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 80px; " src="https://static.igem.org/mediawiki/2014/0/0f/Sall%C3%A8les_d%27Aude.jpg"><br />
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</tr><br />
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<td width="180px"><br />
<img style="width: 70px;" src="https://static.igem.org/mediawiki/2014/d/db/600px-Blason_ville_fr_Ventenac-en-Minervois_%28Aude%29.svg.png"></td><br />
<td colspan="3"><br />
<p class="texte"><br />
Ventenac en Minervois is 600 inhabitants’ village situated in Aude in the Languedoc-Roussillon region (South of France) and the Canal du Midi passes through it.<br />
</p><br />
</td><br />
</tr><br />
<tr><td colspan="4" width="100%" style="padding-top:20px;"><br />
<br />
<br />
<p class="title1">Lab equipment support</p><br />
</td></tr><br />
<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
Thermo Fisher Scientific is one of the leaders in serving science by offering a wide range of innovative technologies and support to make the world cleaner and safer. The company serves pharmaceutical, biotechnology companies as well as hospitals, clinical laboratories.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 130px; " src="https://static.igem.org/mediawiki/2014/2/26/Logo_ThermoFisher.png"><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td width="180px"><br />
<img style="width: 100px;" src="https://static.igem.org/mediawiki/2014/9/91/Qiagen_logo.JPG"><br />
<td colspan="3"><br />
<p class="texte"><br />
Qiagen was created in 1984 and is nowadays a leader in innovative market and technology by creating assay technologies that can enable the access to content from any biological sample. The main purpose is to help achieving success and major breakthroughs in life sciences. Qiagen offers a broad range of 500 products for over 500, 000 customers worldwide.<br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td colspan="3"><br />
<p class="texte"><br />
New Englands Biolabs (NEB) is the leading industry in discovery and production of enzymes and the offer of recombinant and native enzymes since 1970. NEB carried on expanding its product in PCR fields but also gene expression, RNA and cellular analysis. The company is mostly focused on promoting new technologies on the market.<br />
</p><br />
</td><br />
<td width="180px"><br />
<img style="width: 130px; " src="https://static.igem.org/mediawiki/2014/9/9f/Logo_NEB.jpg"><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td width="180px"><br />
<img style="width: 150px;" src="https://static.igem.org/mediawiki/2014/6/60/Eurofins_new.jpg"><br />
<td colspan="3"><br />
<p class="texte"><br />
Eurofins Scientific is a French company created in 1987 and mostly specialized in bioanalysis with more than 100,000 analysis methods and technologies. This industry has an international network of 190 laboratories in 36 different countries with 15 000 employees and has many fields of specialization: food market, environment, pharmaceutical and medical industries, agroscience… Eurofins Scientific contributes to the well-being and the health of everyone by giving high-quality policy advice and analysis services.<br />
</p><br />
</td><br />
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</table><br />
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<br />
<br />
<p class="texte"><br />
If you are interested in any kind of partnership with the Toulouse iGEM team 2014, please contact us at <a href="mailto:igemtoulouse2014@gmail.com">igemtoulouse2014@gmail.com</a><br />
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{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/Acknowledgements/AttributionsTeam:Toulouse/Acknowledgements/Attributions2014-10-17T22:17:46Z<p>Truan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Acknowlegdements&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Attributions</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Ulule</a></li><br />
<li><a href="#select2">Scientists</a></li><br />
<li><a href="#select3">Press</a></li><br />
<li><a href="#select4">Ethics</a></li><br />
<li><a href="#select5">Design and Communication</a></li><br />
<li><a href="#select6">Special thanks</a></li><br />
<li><a href="#select7">Premises</a></li><br />
<li><a href="#select8">Us</a></li><br />
</ul><br />
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<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte">Our team is proud to say that we almost did all the work on our own: finding sponsors, making communication tools about our project, finding the protocols to use and optimize them. However, we needed occasional assistance during our project, therefore we required help from different people: scientists, administrators, specialists and even lay persons. We would like to warmly thank all these persons for their contribution and support. Our project moved forward because they offered their help.</p><br />
<br />
<p class="title1" id="select1">Ulule: a crowdfunding website</p><br />
<br />
<p class="texte">Ulule is the first European crowdfunding website. We have posted our SubtiTree project on this site to collect funds for paying the Giant Jamboree fee. The collect was successful: we had 53 supporters and € 1,787 collected!</br></br><br />
We warmly thank all the supporters, it was very encouraging to see that people mobilized for us!</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/43/Ulule_result.png" width="700px"></center><br />
<br><br />
<p class="texte">We would like to thank:</p><br />
<br />
<p class="nomsd">Debra Baker, François Barbeau, Valérie Chataigner, Brune Couffignal, Serge Couffignal, Jacques de Grenier, Guillaume Debar, Colas Decloitre, Caroline Delabre, Charlene Douard, Laurence Fournié, Rose Fournié, Chrystelle Gaucher, Nicolas Gilet, Aurélie Gosseau, Sarah Guiziou, Carole Jouve, Damien Kanitzer, Pierre Lagoutte, Anne-Marie Lang, Myriam Le Nours, Julie Mabille, Mylène Manière, Eddy Manière, Stéphanie Marty, Nathan Mirassou, Pierre Mirassou, Sophie Molina, Raymond Molina, Eric Molina, Ngoc Thu Hang Pham, Bruno Paci, Frédéric Paletou, Nicolas Perez, Jean Perron, Pierre Pettera, Lucas Picault, Nathalie Pineau, Elodie Prattico, Florence Rigaud, Marianne et Dominique Serres, Gilles Truan, Clémence Witzmann.</p><br />
<br />
<p class="title1" id="select2">Scientists<br />
</p><br />
<p class="title2">iGEM instructors<br />
</p><br />
<p class="texte">First of all, we warmly thank our amazing instructor: <b>Gilles Truan</b>. He was with us from the beginning to the end and even more! He is the master of cloning. He is the captain of PCR. Moreover, he knows how to grill sausages better than anyone!<br />
We kindly thank all the other instructors that helped us when we were lost with the scientific part or stressed by the lack of money. We especially thank <b>Brice Enjalbert</b>, <b>Florence Bordes</b>, <b>Kaymeuang Cam</b>, <b>Magali Remaud-Siméon</b>, <b>Philippe Soucaille</b>, <b>Stéphane Guillouet</b>, <b>Isabelle Meynial-Salles</b> and <b>Alain Marty</b>. Your advice and encouragements were very precious to us, and you deserve these thanks for all the energy you spent answering our questions (and we know that we are very energy-consuming!).<br />
To finish, a special thanks to <b>Claude Maranges</b>, who helped us a lot with the sponsoring aspect and all the administrative problems that we encountered. Indeed, if you have any administrative issue, he is able to solve it in less than 2 minutes (5 minutes during the holidays)! He knows how to make everything smooth in record time.<br />
</p><br />
<br />
<p class="title2"><i>Bacillus subtilis</i> specialists<br />
</p><br />
<p class="texte">Even if <i>Bacillus subtilis</i> is a gram-positive bacterium model, none of our instructors ever worked with it. It is why we asked other people's help when we had questions about <i>B. subtilis</i>.<br />
We would like to thank <b>Nathalie Campo</b> for her advice about <i>B. subtilis</i> culture, her encouragements and her kindness.<br />
Her friend <b>Thierry Doan</b> helped us too: he brilliantly answered our questions about <i>B. subtilis</i>.<br />
We also thank <b>Sarah Guiziou</b>, iGEMer of the 2013 Toulouse team, for her advice and her protocols.<br />
</p><br />
<br />
<p class="title2">Fungi specialists<br />
</p><br />
<p class="texte"><br />
We are grateful to <b>Christophe Roux</b> who gave us two non-pathogenic strains of fungi: <i>Aspergillus brasiliensis</i>, <i>Trichoderma reesei</i>, <i>Aspergillus nidulans</i> and <i>Chaetomium globusum</i>. We used them to test our SubtiTree system. We deeply thank <b>Sylvain Raffaele</b> and <b>Marielle Barascud</b> for their ENORMOUS help at the end of the project. They took time to inject Subtitree in <i>Nicotiana Benthamiana</i>, setting up the ultimate and most valuable proof for our system (see Results). May the force be with them forever...<br />
</p><br />
<br />
<p class="title2">Other iGEM teams<br />
</p><br />
<p class="texte"><br />
Since several parts that we wanted to use were not in the iGEM kit plates, we asked iGEM teams to send us some parts:</br><br />
- Thanks to the <b>iGEM Warsaw team</b>, especially Radoslaw Stachowiak, for sending us the biobricks <br />
<a href="http://parts.igem.org/Part:BBa_K780000" target="_blank">BBa_K780000</a>, <a href="http://parts.igem.org/Part:BBa_K780001" target="_blank">BBa_K780001</a>, <a href="http://parts.igem.org/Part:BBa_K780002" target="_blank">BBa_K780002</a> and <a href="http://parts.igem.org/Part:BBa_K780003" target="_blank">BBa_K780003</a>.</br><br />
- Thanks to the <b>iGEM Utah State team</b>, especially Charles Miller, for sending us the biobrick <a href="http://parts.igem.org/Part:BBa_K1162001" target="_blank">BBa_K1162001</a>.</br><br />
- Thanks to the <b>iGEM Munich team</b>, especially Jara Radek, for sending us the biobrick <a href="http://parts.igem.org/Part:BBa_K823021" target="_blank">BBa_K823021</a>, <a href="http://parts.igem.org/Part:BBa_K823022" target="_blank">BBa_K823022</a> and <a href="http://parts.igem.org/Part:BBa_K823023" target="_blank">BBa_K823023</a>.<br />
</p><br />
<br />
<p class="title2">Lab support<br />
</p><br />
<p class="texte"><br />
Thanks to <b>Sylvie Cancel</b> and <b>Yves Dutruy</b>, lab technicians, for helping us when we did not know where to find Yeast Extract or how to use the new refrigerated centrifuge. They were with us as soon as we began the manipulations and they answer to all our questions. </br><br />
A special thank to <b>Patrick Chekroun</b>, talended glass blower, for making the tubes system used in Chemotaxis tests (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Experimental results</a>.<br><br />
To finish, thanks to <b>Fabien Albert</b>, storeman of the LISBP, for his sympathy and his availability.<br />
</p><br />
<br />
<p class="title1" id="select3">Press<br />
</p><br />
<p class="texte">A lot of different local newspapers were interested in SubtiTree.<br />
In this part, we would like to thank all the journalists that came to meet us:</br><br />
- <b>Hélène Ménal</b> for her article in the newspaper <br />
<a href="http://www.20minutes.fr/toulouse/1416215-20140708-guerisseurs-platanes" target="_blank">20 Minutes</a></br><br />
- <b>Philippe Font</b> for his article in the newspaper <br />
<a href="http://www.metronews.fr/toulouse/ils-etudient-une-bacterie-pour-soigner-les-platanes-du-canal-du-midi/mngj!FOMnxoEasL2s/" target="_blank">Metronews</a></br><br />
- <b>Bernard Davodeau</b> for his article in the newspaper <br />
<a href="http://www.ladepeche.fr/article/2014/07/15/1918689-un-espoir-de-traitement-pour-les-platanes-du-canal.html" target="_blank">La Dépêche</a></br><br />
- <b>Delphine Russeil</b> for her article in the newspaper La Voix du Midi</br><br />
- <b>Angélique Mangon</b> and <b>Jack Levé</b> for their television report on the channel <br />
<a href="http://france3-regions.francetvinfo.fr/midi-pyrenees/2014/07/09/des-etudiants-toulousains-developpent-une-bacterie-pour-sauver-les-platanes-514659.html" target="_blank">France 3 Midi-Pyrénées</a></br><br />
- <b>Philip Hemme</b> for his article about the French iGEM teams on the website <br />
<a href="http://labiotech.fr/les-equipes-francaise-lassaut-digem-2014-competition-mondiale-biologie-synthetique/" target="_blank">LaBiotech.fr</a></br><br />
- <b>Olivier Schlama</b> for his article in the newspaper Midi Libre</br><br />
- <b>Virginie Brancotte</b> for her article in the newspaper <br />
<a href="http://www.fluvialnet.com/murmures-actualites-des-etudiants-toulousains-inventent-une-methode-de-lutte-contre-le-chancre-colore/9576" target="_blank">Fluvial</a><br />
</p><br />
<br />
<p class="title1" id="select4">Ethics<br />
</p><br />
<p class="texte">We thank <b>Vincent Grégoire-Delory</b>, responsible of the ethics platform at Toulouse White Biotechnology, for coming to our lab and sharing his huge knowledge with us around pizzas and a good bottle of wine! He suggested us several points to deepen our ethical reflection.<br />
</p><br />
<br />
<p class="title1" id="select5">Design and Communication<br />
</p><br />
<p class="texte">We thank the <b>INSA Toulouse Communication Department</b> for giving us INSA Toulouse goodies and helping us with the organization of our acknowledgment day.<br><br />
Thanks to <b>Hélène Cabanac</b>, graphic designer, for her help with the design of our presentation and our poster.<br><br />
Thanks to the web designer <b>Adrien Nicod</b> for accepting to work with us and being patient with the remarks that each team member was saying!</p><br />
<br />
<p class="title1" id="select6">Special thanks<br />
</p><br />
<p class="texte">We warmly thank <b>Didier Combes</b>, <b>Nick Lindley</b> , <b>Pierre Monsan</b> and <b>Nicolas Combébiac</b> for their support and their efficiency in the face of our administrative problems.</br><br />
A special thanks to <b>Gilbert Chauvel</b> for his interest into our project and his valuable support.</br><br />
A special thanks to <b>Matthieu Arlat</b> for giving us a lot of good advice, helping us with the sponsoring part and his enthusiasm.</br><br />
To finish, thanks to the God of Synthetic Biology for giving us such results!<br />
</p><br />
<br />
<p class="title1" id="select7">Premises<br />
</p><br />
<p class="texte">We would like to thank the <b>INSA de Toulouse</b> for hosting us during the whole summer (and even more actually!).<br />
</p><br />
<br />
<p class="title1" id="select8">Us<br />
</p><br />
<p class="texte">And finally, we thank... <b>us</b>. For being in the lab all the summer, for our devotion to the plane trees, for the perseverance in the bad days and for putting up each other.</br><br />
Therefore thanks to the entire <a href="https://2014.igem.org/Team:Toulouse/Team">team</a> for this wonderful and exciting project!</br><br />
We would like to thank also our mums and dads because without them we would not be there and thus the project would not have been done! And also the mum and dad of our mums and dads and the mum and dad of the mum and dad of our mums and dads and... you should get it!!<br />
</p><br />
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<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/Acknowledgements/AttributionsTeam:Toulouse/Acknowledgements/Attributions2014-10-17T22:12:21Z<p>Truan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Acknowlegdements&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Attributions</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Ulule</a></li><br />
<li><a href="#select2">Scientists</a></li><br />
<li><a href="#select3">Press</a></li><br />
<li><a href="#select4">Ethics</a></li><br />
<li><a href="#select5">Design and Communication</a></li><br />
<li><a href="#select6">Special thanks</a></li><br />
<li><a href="#select7">Premises</a></li><br />
<li><a href="#select8">Us</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte">Our team is proud to say that we almost did all the work on our own: finding sponsors, making communication tools about our project, finding the protocols to use and optimize them. However, we needed occasional assistance during our project, therefore we required help from different people: scientists, administrators, specialists and even lay persons. We would like to warmly thank all these persons for their contribution and support. Our project moved forward because they offered their help.</p><br />
<br />
<p class="title1" id="select1">Ulule: a crowdfunding website</p><br />
<br />
<p class="texte">Ulule is the first European crowdfunding website. We have posted our SubtiTree project on this site to collect funds for paying the Giant Jamboree fee. The collect was successful: we had 53 supporters and € 1,787 collected!</br></br><br />
We warmly thank all the supporters, it was very encouraging to see that people mobilized for us!</p><br />
<center><img src="https://static.igem.org/mediawiki/2014/4/43/Ulule_result.png" width="700px"></center><br />
<br><br />
<p class="texte">We would like to thank:</p><br />
<br />
<p class="nomsd">Debra Baker, François Barbeau, Valérie Chataigner, Brune Couffignal, Serge Couffignal, Jacques de Grenier, Guillaume Debar, Colas Decloitre, Caroline Delabre, Charlene Douard, Laurence Fournié, Rose Fournié, Chrystelle Gaucher, Nicolas Gilet, Aurélie Gosseau, Sarah Guiziou, Carole Jouve, Damien Kanitzer, Pierre Lagoutte, Anne-Marie Lang, Myriam Le Nours, Julie Mabille, Mylène Manière, Eddy Manière, Stéphanie Marty, Nathan Mirassou, Pierre Mirassou, Sophie Molina, Raymond Molina, Eric Molina, Ngoc Thu Hang Pham, Bruno Paci, Frédéric Paletou, Nicolas Perez, Jean Perron, Pierre Pettera, Lucas Picault, Nathalie Pineau, Elodie Prattico, Florence Rigaud, Marianne et Dominique Serres, Gilles Truan, Clémence Witzmann.</p><br />
<br />
<p class="title1" id="select2">Scientists<br />
</p><br />
<p class="title2">iGEM instructors<br />
</p><br />
<p class="texte">First of all, we warmly thank our amazing instructor: <b>Gilles Truan</b>. He was with us from the beginning to the end and even more! He is the master of cloning. He is the captain of PCR. Moreover, he knows how to grill sausages better than anyone!<br />
We kindly thank all the other instructors that helped us when we were lost with the scientific part or stressed by the lack of money. We especially thank <b>Brice Enjalbert</b>, <b>Florence Bordes</b>, <b>Kaymeuang Cam</b>, <b>Magali Remaud-Siméon</b>, <b>Philippe Soucaille</b>, <b>Stéphane Guillouet</b>, <b>Isabelle Meynial-Salles</b> and <b>Alain Marty</b>. Your advice and encouragements were very precious to us, and you deserve these thanks for all the energy you spent answering our questions (and we know that we are very energy-consuming!).<br />
To finish, a special thanks to <b>Claude Maranges</b>, who helped us a lot with the sponsoring aspect and all the administrative problems that we encountered. Indeed, if you have any administrative issue, he is able to solve it in less than 2 minutes (5 minutes during the holidays)! He knows how to make everything smooth in record time.<br />
</p><br />
<br />
<p class="title2"><i>Bacillus subtilis</i> specialists<br />
</p><br />
<p class="texte">Even if <i>Bacillus subtilis</i> is a gram-positive bacterium model, none of our instructors ever worked with it. It is why we asked other people's help when we had questions about <i>B. subtilis</i>.<br />
We would like to thank <b>Nathalie Campo</b> for her advice about <i>B. subtilis</i> culture, her encouragements and her kindness.<br />
Her friend <b>Thierry Doan</b> helped us too: he brilliantly answered our questions about <i>B. subtilis</i>.<br />
We also thank <b>Sarah Guiziou</b>, iGEMer of the 2013 Toulouse team, for her advice and her protocols.<br />
</p><br />
<br />
<p class="title2">Fungi specialists<br />
</p><br />
<p class="texte"><br />
We are grateful to <b>Christophe Roux</b> who gave us two non-pathogenic strains of fungi: <i>Aspergillus brasiliensis</i>, <i>Trichoderma reesei</i>, <i>Aspergillus nidulans</i> and <i>Chaetomium globusum</i>. We used them to test our SubtiTree system.<br />
</p><br />
<br />
<p class="title2">Other iGEM teams<br />
</p><br />
<p class="texte"><br />
Since several parts that we wanted to use were not in the iGEM kit plates, we asked iGEM teams to send us some parts:</br><br />
- Thanks to the <b>iGEM Warsaw team</b>, especially Radoslaw Stachowiak, for sending us the biobricks <br />
<a href="http://parts.igem.org/Part:BBa_K780000" target="_blank">BBa_K780000</a>, <a href="http://parts.igem.org/Part:BBa_K780001" target="_blank">BBa_K780001</a>, <a href="http://parts.igem.org/Part:BBa_K780002" target="_blank">BBa_K780002</a> and <a href="http://parts.igem.org/Part:BBa_K780003" target="_blank">BBa_K780003</a>.</br><br />
- Thanks to the <b>iGEM Utah State team</b>, especially Charles Miller, for sending us the biobrick <a href="http://parts.igem.org/Part:BBa_K1162001" target="_blank">BBa_K1162001</a>.</br><br />
- Thanks to the <b>iGEM Munich team</b>, especially Jara Radek, for sending us the biobrick <a href="http://parts.igem.org/Part:BBa_K823021" target="_blank">BBa_K823021</a>, <a href="http://parts.igem.org/Part:BBa_K823022" target="_blank">BBa_K823022</a> and <a href="http://parts.igem.org/Part:BBa_K823023" target="_blank">BBa_K823023</a>.<br />
</p><br />
<br />
<p class="title2">Lab support<br />
</p><br />
<p class="texte"><br />
Thanks to <b>Sylvie Cancel</b> and <b>Yves Dutruy</b>, lab technicians, for helping us when we did not know where to find Yeast Extract or how to use the new refrigerated centrifuge. They were with us as soon as we began the manipulations and they answer to all our questions. </br><br />
A special thank to <b>Patrick Chekroun</b>, talended glass blower, for making the tubes system used in Chemotaxis tests (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Experimental results</a>.<br><br />
To finish, thanks to <b>Fabien Albert</b>, storeman of the LISBP, for his sympathy and his availability.<br />
</p><br />
<br />
<p class="title1" id="select3">Press<br />
</p><br />
<p class="texte">A lot of different local newspapers were interested in SubtiTree.<br />
In this part, we would like to thank all the journalists that came to meet us:</br><br />
- <b>Hélène Ménal</b> for her article in the newspaper <br />
<a href="http://www.20minutes.fr/toulouse/1416215-20140708-guerisseurs-platanes" target="_blank">20 Minutes</a></br><br />
- <b>Philippe Font</b> for his article in the newspaper <br />
<a href="http://www.metronews.fr/toulouse/ils-etudient-une-bacterie-pour-soigner-les-platanes-du-canal-du-midi/mngj!FOMnxoEasL2s/" target="_blank">Metronews</a></br><br />
- <b>Bernard Davodeau</b> for his article in the newspaper <br />
<a href="http://www.ladepeche.fr/article/2014/07/15/1918689-un-espoir-de-traitement-pour-les-platanes-du-canal.html" target="_blank">La Dépêche</a></br><br />
- <b>Delphine Russeil</b> for her article in the newspaper La Voix du Midi</br><br />
- <b>Angélique Mangon</b> and <b>Jack Levé</b> for their television report on the channel <br />
<a href="http://france3-regions.francetvinfo.fr/midi-pyrenees/2014/07/09/des-etudiants-toulousains-developpent-une-bacterie-pour-sauver-les-platanes-514659.html" target="_blank">France 3 Midi-Pyrénées</a></br><br />
- <b>Philip Hemme</b> for his article about the French iGEM teams on the website <br />
<a href="http://labiotech.fr/les-equipes-francaise-lassaut-digem-2014-competition-mondiale-biologie-synthetique/" target="_blank">LaBiotech.fr</a></br><br />
- <b>Olivier Schlama</b> for his article in the newspaper Midi Libre</br><br />
- <b>Virginie Brancotte</b> for her article in the newspaper <br />
<a href="http://www.fluvialnet.com/murmures-actualites-des-etudiants-toulousains-inventent-une-methode-de-lutte-contre-le-chancre-colore/9576" target="_blank">Fluvial</a><br />
</p><br />
<br />
<p class="title1" id="select4">Ethics<br />
</p><br />
<p class="texte">We thank <b>Vincent Grégoire-Delory</b>, responsible of the ethics platform at Toulouse White Biotechnology, for coming to our lab and sharing his huge knowledge with us around pizzas and a good bottle of wine! He suggested us several points to deepen our ethical reflection.<br />
</p><br />
<br />
<p class="title1" id="select5">Design and Communication<br />
</p><br />
<p class="texte">We thank the <b>INSA Toulouse Communication Department</b> for giving us INSA Toulouse goodies and helping us with the organization of our acknowledgment day.<br><br />
Thanks to <b>Hélène Cabanac</b>, graphic designer, for her help with the design of our presentation and our poster.<br><br />
Thanks to the web designer <b>Adrien Nicod</b> for accepting to work with us and being patient with the remarks that each team member was saying!</p><br />
<br />
<p class="title1" id="select6">Special thanks<br />
</p><br />
<p class="texte">We warmly thank <b>Didier Combes</b>, <b>Nick Lindley</b> , <b>Pierre Monsan</b> and <b>Nicolas Combébiac</b> for their support and their efficiency in the face of our administrative problems.</br><br />
A special thanks to <b>Gilbert Chauvel</b> for his interest into our project and his valuable support.</br><br />
A special thanks to <b>Matthieu Arlat</b> for giving us a lot of good advice, helping us with the sponsoring part and his enthusiasm.</br><br />
To finish, thanks to the God of Synthetic Biology for giving us such results!<br />
</p><br />
<br />
<p class="title1" id="select7">Premises<br />
</p><br />
<p class="texte">We would like to thank the <b>INSA de Toulouse</b> for hosting us during the whole summer (and even more actually!).<br />
</p><br />
<br />
<p class="title1" id="select8">Us<br />
</p><br />
<p class="texte">And finally, we thank... <b>us</b>. For being in the lab all the summer, for our devotion to the plane trees, for the perseverance in the bad days and for putting up each other.</br><br />
Therefore thanks to the entire <a href="https://2014.igem.org/Team:Toulouse/Team">team</a> for this wonderful and exciting project!</br><br />
We would like to thank also our mums and dads because without them we would not be there and thus the project would not have been done! And also the mum and dad of our mums and dads and the mum and dad of the mum and dad of our mums and dads and... you should get it!!<br />
</p><br />
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Notebook&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Protocols</p> <br />
<ul class="topnav" id="topnav" style="top:15px;"><br />
<br />
</ul><br />
</div><br />
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<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li><br />
<li><a href="#select2"><i>E. coli</i> transformation protocol</a></li><br />
<li><a href="#select3">Miniprep and alcaline lysis</a></li><br />
<li><a href="#select4">Cloning</a></li><br />
<li><a href="#select5">Checking of the genetic constructions</a></li><br />
<li><a href="#select6"><i>B. subtilis</i> transformation</a></li><br />
<li><a href="#select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</a></li><br />
<li><a href="#select8">Final Tests</a></li><br />
</ul><br />
</div><br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<p class="texte"> All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...). Finally they gave us some <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">results</a> :-).</p><br />
<br />
<p class="title1" id="select1"><I>E. coli</I> competent cells</p><br />
<p class="texte"><I> <CENTER> MANIPULATION IN ICE </CENTER> </I></p><br />
<p class="texte"><B> Day 0 </B><br />
<br>- Make an <i>Escherichia coli</i> cell culture in LB medium overnight<br />
<p><br />
<p class="texte"><B> Day 1 </B><br />
<br/> <br />
- Freeze 0.1M CaCl<sub>2</sub> and 4 Falcon tubes of 50mL at 4°C<br />
<br/> <br />
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)<br />
<br/> <br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Remove the supernatant<br />
<br/><br />
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub> <br />
<br/><br />
- Centrifuge 10 minutes at 4500 RPM<br />
<br/><br />
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub><br />
<br/><br />
- Add glycerol to a final concentration of 15%<br />
<br/><br />
- Keep the tubes at -80°C<br />
</p><br />
<br />
<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p><br />
<p class="texte"><br />
- Let the LB agar medium plates dry in a sterile area<br />
<br/><br />
- Thaw out the competent cell aliquotes for about 10 to 20 minutes<br />
<br/><br />
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA <br />
<br/><br />
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i><br />
- Put the tubes 20 minutes in the ice<br />
<br/><br />
- Put the tubes 2 minutes at 42°C in the water bath<br />
<br/><br />
- Put the tubes back in ice immediately to create the thermic shock<br />
<br/><br />
- Add 1mL of LB medium<br />
<br/><br />
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression<br />
<br/><br />
- Centrifuge for 1 minute at 13000 RPM<br />
<br/><br />
- Remove the supernatant <br />
<br/><br />
- Resuspend in 250 µL of LB medium<br />
<br/><br />
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate<br />
</p><br />
<br />
<p class="title1" id="select3"> Miniprep and alcaline lysis </p><br />
<p class="texte"><B> Day 0 </B><br />
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)<br />
<br/><br />
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic<br />
<br/><br />
- Let the culture grow overnight at 37°C in a shaking incubator</p> <br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br>- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C.<br />
<br/><br />
- Keep the tubes at -20°C <br />
<br><br />
<br />
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.<br />
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. <br />
<br><br />
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM<br />
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%<br />
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid<br />
</I></p><br />
<br />
<p class="title1" id="select4"> Cloning </p><br />
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.<br />
<br><br />
<p class="title2">First step</p><br />
<p class="title3">Both parts have the same antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b><br />
<br> For the vector :<br />
<br>- 5 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 9 µL of Milli-Q water <br />
<br><br />
<br> For the insert :<br />
<br>- 10 µL of miniprep plasmid <br />
<br><br />
- 2 µL of each restriction enzymes<br />
<br><br />
- 2 µL of Green Buffer<br />
<br><br />
- 4 µL of Milli-Q water <br />
<br><br />
- Incubate 15 minutes at 37°C <br />
<br />
<br><br><b>2) Gel extraction</b><br />
<br><br />
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer<br />
<br><br />
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well<br />
<br><br />
- Migration for 30 min at 100 V or 1 hour at 50V<br />
<br><br />
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes<br />
<br><br />
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit<br />
<br />
<br><br>3) Inactivation of the enzymes for the vector<br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title3">The two parts have a different antibiotic resistance</p><br />
<p class="texte"><b>1) Digestion mix</b> <br />
<br>For each part, add: <br />
<br>- 5 µL of miniprep plasmid <br />
<br>- 1 µL of each restriction enzymes<br />
<br>- 2 µL of Green Buffer<br />
<br>- 9 µL of Milli-Q water <br />
<br>- Incubate 15 minutes at 37°C <br />
<br />
<p class="texte"><br />
<b>2) Inactivation of the enzymes for the vector</b><br />
<br>There are two ways to inactivate the enzymes:<br />
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb<br />
<br>- Heat inactivation at 95°C for 10 minutes.</p><br />
<br />
<p class="title2">Second step</p><br />
<p class="title3">Ligation</p><br />
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water<br />
<br/><br />
A control without insert must be made<br />
<br/><br />
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation</p><br />
<br />
<p class="title3">Transformation</p><br />
<p class="texte"><br />
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.<br />
<br/><br />
- Plate the solution on selective medium overnight at 37°C.</p><br />
<br />
<p class="title1 " id="select5">Checking of the genetic constructions </p><br />
<p class="title2">1) Colony PCR</p><br />
<p class="texte"><br />
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.<br />
<br/><br />
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.<br />
<br/>The following cycles have been used : <br />
<br/>- 94°C - 5 min<br />
<br/>- (94°C 45 sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles <br />
<br/>- 72°C - 5min<br />
<br/>- Then 4°C</p><br />
<br />
<p class="title2">2) Analytic digestion</p><br />
<p class="texte"><br />
- Put a colony in 5 mL of LB selective medium and wait for 6 hours<br />
<br/><br />
- Make a purification thanks to the Miniprep kit<br />
<br/><br />
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL<br />
<br/><br />
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p><br />
<br />
<p class="title2">3) Sequencing</p><br />
<p class="texte"><br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.</p><br />
<br />
<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p><br />
<br />
<p class="texte"><br />
<B> Day 0 </B><br />
<br/>- Streak out the <i>Bacillus</i> strain and plate this on an LB agar plate overnight at 37°C</p><br />
<br />
<p class="texte"><B> Day 1 </B><br />
<br />
<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2ml of completed 1x MC<br />
<br/><br />
- Grow at 37°C for 5 hours<br />
<br/><br />
- Mix 400µl of culture in a fresh tube (tubes loosely closed for the aeration) and put 5µL of Miniprep DNA.<br />
<br/><br />
- Grow the cells at 37°C for an additional 2 hours<br />
<br/><br />
- Spread the complete 400 µl reaction mix on selective antibiotic plates (100µl per plate), and incubate at 37°C overnight <br />
<br/></p><br />
<br />
<p class="texte"><b> Preparation of solutions: </b><br><br />
<I> <br> 300 mM Tri-Na Citrate:</I><br />
<br>- 0.88 g Tri-Na Citrate<br />
<br>- 10mL MQ water<br />
<br><br><I>Ferric NH4 citrate:</I><br />
<br>- 0.22g Ferric NH4<br />
<br>- 10mL MQ water<br />
<br><br><I>10x Competence Medium </I><br />
<br> For 10mL:<br />
<br>- 1.40g K2HPO4<br />
<br>- 0.52g KH2PO4<br />
<br>- 2g glucose<br />
<br>- 1 mL 300 mM Tri-Na citrate <br />
<br>- 0.1 mL Ferric NH4 citrate<br />
<br>- 0.1g Casein Hydrolysate<br />
<br>- 0.2 g Potassium glutamate<br />
<br>The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br><br><I>1x Competence Medium </I><br />
<br>- 1.8 mL MQ water<br />
<br>- 200 µL 10x Competence Medium solution (previously filter sterilized)<br />
<br>- 6.7 µL 1M MgSO4 (previously autoclaved)<br />
<br>- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)<br />
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.<br />
<br />
<br />
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p><br />
<p class="texte"><br />
<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight <br />
<br>- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin. <br />
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.<br />
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.<br />
<center><img src="https://static.igem.org/mediawiki/2014/a/a3/Thr.png" width="400px"></center><br />
<p class="legend">Threnonine test (Left: MC Thr+; Right: MC Thr -)</p><br />
<br />
<p class="title1" id="select8">Final Tests</p><br />
<p class="title2">Chemotaxis test</p><br />
<p class="texte"><br />
Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the <a href="https://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">« Capillary essay »</a> from Imperial College 2011 iGEM team.<br />
<br>- Prepare the bacteria in LB medium until they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.<br />
<br>- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2014/b/b1/Installation_1.gif" width="650px"></center><br />
<p class="texte"><br />
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br><br />
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.<br> <br />
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.<br />
<br>- Let the installation settle for 1 hour at room temperature.<br />
<br>- After an hour, put the volume of the tips on parafilm. <br />
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.<br />
<br>- The plates are then incubated overnight at 37°C.</p><br />
<br />
<p class="title2">Binding test</p><br />
<p class="texte"><i>CBB (Chitin Binding Buffer):</i><br />
<br>- 500 mM NaCl<br />
<br>- 20 mM Tris-HCl<br />
<br>- 1 mM EDTA<br />
<br>- 0,05% Triton X-100, 25°C, pH=8<br />
</p><br />
<br />
<p class="title3">Column activation:</p><br />
<p class="texte">- Vortex the beads <br />
<br>- Put 50 µL of beads in a 1.5mL centrifuge tube<br />
<br>- Wash with 500 µL of CBB<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Repeat the wash<br />
</p><br />
<br />
<br />
<p class="title3">Bacterial fixation on the chitin beads:</p><p class="texte"><br />
- Add 200 µL of bacteria solution (10<sup>5</sup> bacteria/mL) to the washed beads <br />
<br>- Shake during 1h at 4°C<br />
<br>- Add 500µL of CBB (washing A)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB (washing B)<br />
<br>- Put the centrifuge tube on a magnetic rack<br />
<br>- Wait 30 seconds<br />
<br>- Remove supernatant<br />
<br>- Add 500µL of CBB to recover the beads directly<br />
</p><br />
<br />
<p class="title3">Bacteria count:</p><br />
<p class="texte">- Make different dilutions : 10<sup>-1</sup>, 10<sup>-3</sup>, 10<sup>-5</sup> of the first bacterial culture and spread on LA plates<br />
<br>- Make different dilutions : 1, 10<sup>-2</sup>, 10<sup>-4</sup> of washings (A and B) and of the beads in CBB medium and spread on LA plates<br />
<br>- Place the plates at 37°C overnight<br />
<br>- Count colonies on different plates<br />
</p><br />
<br />
<p class="title2">Fungicide test: anti-fungal activities</p><br />
<p class="texte"><br />
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br><br />
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i><br />
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.<br />
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.<br />
<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.<br />
<br><i>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.</i><br />
<br>- After 72 hours of liquid culture of the different clones of <i>B. subtilis</i> with the fungicides module, the culture can be centrifugated.<br />
<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad. <br />
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.<br />
</p><br />
<br />
<p class="title2">Fungicide test: <i>in planta</i> assay</p><br />
<p class="title3">First step</p><br />
<p class="texte"><br />
The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). <br>We diluted our bacterial samples to get two concentrations: 5.10<sup>6</sup> and 10<sup>8</sup> bacteria per mL. The WT and transformed bacteria are introduced into plants (a control test without bacteria is performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Bacterial growth in the plant is left for 24 h.<br />
</p><br />
<p class="title3">Second step</p><br />
<p class="texte"><br />
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.<br />
</p><br />
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{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/CommunicationTeam:Toulouse/Communication2014-10-17T21:44:24Z<p>Truan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Communication</p> <br />
</div><br />
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Press</a></li><br />
<li><a href="#select2">Interventions</a></li><br />
<li><a href="#select3">Interactions with other iGEM teams</a></li><br />
<li><a href="#select4">Newsletters</a></li><br />
<li><a href="#select5">Communication tools</a></li><br />
</ul><br />
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<br />
<p class="citation"> <br />
"Good words are worth much, and cost little."</p><br />
<br />
<!--Press--><br />
<p class="title1" id="select1">Press</p><br />
<p class="texte">As our project was built on a regional problem, a lot of different local newspapers were interested into it.<br><br />
After the first articles, other newspapers have begun to be interested into our work. It helped us to have a better visibility.<br><br />
We have listed all the articles :<br><br />
<center><table width="80%"><br />
<tr><td><p class="texte"><br />
- <a href="http://www.20minutes.fr/toulouse/1416215-20140708-guerisseurs-platanes">20Minutes</a><br><br />
- <a href="http://www.metronews.fr/toulouse/ils-etudient-une-bacterie-pour-soigner-les-platanes-du-canal-du-midi/mngj!FOMnxoEasL2s/">Metronews</a><br><br />
- <a href="http://www.ladepeche.fr/article/2014/07/15/1918689-un-espoir-de-traitement-pour-les-platanes-du-canal.html">LaDépêche</a><br><br />
- La Voix du Midi<br><br />
- <a href="http://labiotech.fr/les-equipes-francaise-lassaut-digem-2014-competition-mondiale-biologie-synthetique/">LaBiotech</a><br><br />
- <a href="http://www.humanite.fr/un-bourgeon-despoir-pour-les-platanes-du-canal-du-midi-548812">L'Humanité</a><br><br />
- <a href="http://www.developpement-durable-networkvisio.com/n31-france/article-toulouse.html?id=10765">Développement durable</a><br><br />
- Midi Libre<br><br />
- <a href="http://www.lindependant.fr/2014/08/15/vnf-aide-la-recherche-pour-sauver-les-platanes,1918464.php">L'Indépendant</a><br><br />
- <a href="http://www.digischool.fr/initiatives/etudiants-veulent-sauver-platanes-canal-du-midi-23990.php">digiSchool</a><br><br />
- La Voix du Midi Lauragais<br><br />
- <a href="http://www.lurioaddl.com/Pages/CanalMidi.aspx">LURIO Addl</a><br><br />
- <a hrf="http://www.fluvialnet.com/murmures-pop-actualites-des-etudiants-toulousains-inventent-une-methode-de-lutte-contre-le-chancre-colore/9576">Fluvial</a><br><br />
- <a hrf="http://www.toulouseblog.fr/actualite-27679-etudiants-toulousains-participent-a-competition-internationale-a-boston.html">Toulouse Blog</a><br><br />
- <a hrf="http://etudiant.lefigaro.fr/flash/flash-actu/detail/article/des-etudiants-toulousains-au-mit-pour-sauver-les-platanes-du-canal-du-midi-8976/">Le Figaro étudiants</a><br></p><br />
</td><br />
<td><img width="200px"; src="https://static.igem.org/mediawiki/2014/1/11/Articles.png"></td></tr><br />
<tr><td><p class="texte"><br />
The Television was also interested in our project : the regional channel <a href="http://france3-regions.francetvinfo.fr/midi-pyrenees/2014/07/09/des-etudiants-toulousains-developpent-une-bacterie-pour-sauver-les-platanes-514659.html">France 3 Midi-Pyrénées</a><br />
came to our lab for an interview.</p></td><br />
<td><img width="250px"; src="https://static.igem.org/mediawiki/2014/3/37/France_3_interview.png"></td></tr><br />
<tr><td><img width="140px"; src="https://static.igem.org/mediawiki/2014/0/0d/France_inter_interview.png"></td><br />
<td><p class="texte">To finish, the national radio France Inter interviewed us in our lab. The interview will be on air when we are in Boston (Dec. 2<sup>nd</sup>!!!</td></tr><br />
</p></table></center><br><br />
<br />
<!--Interventions--><br />
<p class="title1" id="select2">Interventions<br></p><br />
<p class="texte">We participated to a lot of public communications about synthetic biology and our project:</p><br><br />
<br><center><table width="80%"><br />
<br />
<tr><td><p class="texte">- The <b>Agora</b> at the University Paul-Sabatier in Toulouse (22<sup>nd</sup> April 2014). It is a monthly conference set up by the association<br />
<a href="http://www.arborisscientiae.fr/">Arboris Scientiae</a></p></td><br />
<td><img style="width:180px"; src="https://static.igem.org/mediawiki/2014/7/7d/22042014-Agora-PierreFlorie1.jpg"><br>Pierre and Florie during the Agora.</td></tr><br />
<br />
<tr><td><img width="100px"; src="https://static.igem.org/mediawiki/2014/4/4e/Exposciences_Toulouse_2014.jpg"><br>Florie and Fanny during the Exposcience.</td><br />
<td valign="middle"><p class="texte">- The Journées de l’Ecole Doctorale Biologie, Santé, Biotechnologies (17th April 2014). This day was dedicated to the exhibition of PhD's results with posters. <br />
We came with a poster explaining what is the iGEM competition.</td></tr><br />
<br />
<tr><td><p class="texte">- The <b>Exposcience</b> in Toulouse (5<sup>th</sup> june 2014). <br />
We were there during the whole day. <br />
We met hundreds of people and explained our project. <br />
We brought lab stuff and a poster describing the iGEM competition.</p></td><br />
<td valign="middle"><img width="150px"; src="https://static.igem.org/mediawiki/2014/0/00/Exposciences_1.jpg"><br>Manon and Camille during the Exposcience</td></tr><br />
<br />
<tr><td valign="middle"><img width="200px"src="https://static.igem.org/mediawiki/2014/e/e5/Colloque_BioSynSys.jpg"><br>Pierre and Mathieu during the BioSynSys conference.</td><br />
<td valign="middle"><p class="texte">- The <a href="http://biosynsys2014.sciencesconf.org/"><b>BioSynSys conference</b></a> (2<sup>nd</sup> july 2014)<br />
where we made an oral presentation in front of 150 scientists specialized in synthetic biology.</p></td></tr><br />
<br />
<br />
<tr><td><p class="texte">- The <b>Novela</b> in Toulouse (4<sup>th</sup> and 11<sup>th</sup> october 2014) where we gave a small communication and further had a public discussion about synthetic biology and human health.</p></td><br />
<td valign="middle"><img width="200px" src="https://static.igem.org/mediawiki/2014/9/95/Novela.png"><br>Camille and Manon during the Novela.</td></tr><br />
</table></center><br />
</p><br />
<br />
<!--Other iGEM teams--><br />
<p class="title1" id="select3">Interactions with other iGEM teams<br></p><br />
<p class="texte"><br />
- Filling in the iGEM Virginia online survey to gauge acceptance and understanding of synthetic biology by the general public (11<sup>st</sup> July).</p><br><br />
<div id="virginiabadge"><br />
<a href=<br />
"https://2014.igem.org/Team:Virginia/HumanPractices"><img height="180"<br />
src="https://static.igem.org/mediawiki/2014/b/b0/Virginia_Transparent.png"<br />
width="250"></a><br />
</div><br><br />
<p class="texte">- Skype with iGEM Columbia (31<sup>st</sup> July) : they explained their contest "Low Budget". Finally, we did not have the time to take part of this contest.<br><br />
- Skype with iGEM York (29<sup>th</sup> August) : we answered questions about women in science</p><br><br />
<br />
<br />
<!--Newsletter--><br />
<p class="title1" id="select4">Newsletters</p><br />
<p class="texte">We set up a monthly Newsletter to inform our sponsors and other people interested in our project. You can find these here (french version):<br> <br />
- <a href="https://static.igem.org/mediawiki/2014/a/ad/Newsletter_iGEM_n%C2%B01_-_juillet_2014.jpg">Newsletter n°1</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/3/32/Newsletter_iGEM_n%C2%B02_-_ao%C3%BBt_2014.pdf">Newsletter n°2</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/8/8b/Newsletter_iGEM_n%C2%B03_-_septembre_2014.pdf">Newsletter n°3</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/e/e1/Newsletter_iGEM_n%C2%B04_-_octobre_2014.pdf">Newsletter n°4</a><br><br />
</p><br />
<br />
<!--Communication tools--><br />
<p class="title1" id="select5">Communication tools</p><br />
<p class="texte">Here you can find all our communication tools: <br><br />
- <a href="https://2014.igem.org/File:IGEM_plaquette.jpg">iGEM Toulouse presentation flyer (French version)</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/e/e2/Plaquette_SubtiTree.pdf">SubtiTree presentation flyer (French version)</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/d/de/Poster_igem_VF.jpg">iGEM Toulouse presentation poster (French version)</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/e/ed/IGEM_Toulouse_2014_logo.pdf">iGEM Toulouse logo</a><br><br />
- <a href="">iGEM Toulouse Giant Jamboree poster (Coming soon)</a><br><br />
- <a href="">iGEM Toulouse Giant Jamboree presentation (Coming soon)</a><br><br />
</p><br />
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{{:Team:Toulouse/template/footer}}</div>Truanhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T21:32:18Z<p>Truan: </p>
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<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
the famous philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
class=GramE>Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human<br />
intervention on Nature</p><br />
<p class="texte">Our main<br />
question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature<br />
considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observe an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
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<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Safety</p> <br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">iGEM Safety</a></li><br />
<li><a href="#select2">Chassis Organism</a></li><br />
<li><a href="#select3">New and/or modified coding region</a></li><br />
<li><a href="#select4">Team safety</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Safety is not just a slogan, it's a way of life"<br />
</p><br />
<br />
<br><br />
<!--PARTIE iGEM SAFETY--><br />
<p class="title1" id="select1">iGEM Safety</p><br />
<p class ="texte">Our safety form was approved in September 2014 by the iGEM team. We filled it with the help of Nathalie Doubrovine, safety officer at the LISBP.<br />
</p><br />
<br />
<!--TABLE CHASSIS ORGANISM--><br />
<p class="title1" id="select2">Chassis organisms</p><br />
<br />
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><br />
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<br />
<!--1ere ligne--><br />
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<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 30px;">Species name (including strain)</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px;">Risk group</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px;">Risk Group Source</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; padding-top: 42px; font-size: 17px; ">Disease risk for humans ?</td><br />
</tr><br />
<!--2eme ligne--><br />
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<td><i><p class ="texte" style="padding-top: 10px; text-align:center;">Escherichia coli MC1061</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-7140.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Bacillus subtilis 168</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-23778.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Aspergillus brasiliensis 246.65 CBS</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-63263.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Chaetomium globosum 148.51 CBS</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-1962.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Trichoderma reesei CBS 383.78</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-768.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Aspergillus nidulans CBS 124.59</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-820.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
<br><br><br />
<br />
<br />
<!--TABLE NEW AND/OR MODIFIED CODING REGION--><br />
<p class="title1" id="select3">New and/or modified coding region</p><br />
<br />
<table style="border-width:1px; border-color:#ccc;width:100%;background-color:#ccc;"<br />
><br />
<tbody style="background-color:white;"><br />
<br />
<!--1ere ligne--><br />
<tr><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 22px 10px; font-size: 15px;">Part number/name</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">Natural function of part</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">How did you acquire it?</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">How will you use it?</p></td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">N-acetylatedGlucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS - Antifungal GAFP-1</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + N-acetyl-glucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + Chitin Binding Protein for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allow the bacterium to bind to the fungus</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364006">BBa_K1364006</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Double expression cassette</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach and bind to the fungus</p></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS + Antifungal GAFP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</p></td><br />
<td><p class ="texte">P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub>-SpoVG + EcAMP-1</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub>-spoVG + EcAMP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--14eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RFP</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fluorescent protein. Designed to evaluate promotor P<sub>veg</sub> strength.</p></td><br />
</tr><br />
<!--16eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>lepA</sub> + RFP</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fluorescent protein. Designed to evaluate promotor P<sub>lepA</sub> strength.</p></td><br />
</tr><br />
<!--17eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Promotor P<sub>lepA</sub> + RBS spoVG</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">High efficiency promoter and RBS for translation.</p></td><br />
</tr><br />
<!--18eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RBS + Antifungal EcAMP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--19eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Integrative plasmid for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part is an integrative vector for <i>Bacillus subtilis</i>.</p></td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
<br><br />
<br />
<p class="title1" id="select4">Team safety</p><br />
<p class="title2">Safety in INSA Toulouse</p><br />
<p class="texte">INSA Toulouse is a public school for engineers. The biosafety guidelines are not specific to our institution; the French National regulations for working conditions and for manipulating genetically modified organisms are applied.<br><br />
The regulation about the workers' prevention against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994) includes microorganisms, cell cultures and human endoparasites which may cause infections, allergies or toxicity. <br><br />
This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5.)<br><br />
The <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000465273&dateTexte=&categorieLien=id">Decree of the 16th July 2007</a> describes the technical preventive measuresto set up in research laboratories (including containment), education, analysis, anatomy and surgical pathology, autopsy rooms, and industrial and agricultural facilities where workers are likely to be exposed to biological pathogens.<br><br />
The rules of health, safety, and preventive medicine applied in public services in France (and thus in all public facilities working in scientific and technological domains) are set out in the <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=LEGITEXT000006063791">Decree No. 82-453</a>. This decree refers to the Labour Code, Public Health Code and Environmental Code.<br><br />
The <a href="http://legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000024584737&categorieLien=id">Decree No. 2011-1177</a> is related to the use of genetically modified organisms.<br><br />
<br><br />
There is no one in charge of the biological safety at the INSA Toulouse. However there is an organization which includes one prevention advisor and several prevention assistants in every Laboratory. As far as the LISBP (our structure) is concerned, Nathalie Doubrovine is the prevention assistant.<br><br />
We have already discussed our project with her. She has given us the safety training and advices about how to respond to an imminent danger.<br><br />
She raised the problem of GMO and the related policy. She advised us of the special procedure for the destruction of GMOs: we cannot wash this kind of organism down the drain.<br><br />
We were also informed about the potential chemical and microbiological risks.<br><br />
<br><br />
The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedures of the LISBP.<br><br />
The legislation requires employers to inform "all new employees" of the risks that they may encounter as well as ways to protect themselves from those risks. Therefore any newcomer must follow an appropriate training and pass a practical test about safety to ensure its own protection as well as protecting his/her colleagues.<br><br />
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).<br><br />
The training is divided into two parts. The first one is a training concerning general risk prevention in research laboratories. This one is made individually with the NEO software and with the explanations of the prevention assistant. The second one is a training about the techniques used during the occupation.<br><br />
<br><br />
<br />
We also hada training to learn how to sort the different sorts of biological waste and how they are destroyed. We also learned that we cannot use autoclave on our own because a specific training is needed. We did not do this specific training and hence did not touch the autoclave. Only our supervisors could start these autoclaves...<br><br />
</p><br />
<br />
<p class="title2">Safety in the lab</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"If you don't think it's safe, it probably isn't"</p><br />
<p class="texte">We have organized our workspace. We have a relaxing room where no biological material should enter. In this room, we can eat and drink (a fridge, a kettle and a coffeemaker are available) but it is also the room where we have our meetings and where we can work on our computers. On the contrary, in the lab, we have to wear protective equipment and respect basic rules.</p><br />
<br />
<p class="title3">Personal protective equipment</p><br />
<p class="texte">As soon as we manipulate in the lab, we have to wear the following personal protective equipment:</p><br />
<table><br />
<tr><td><p class="texte">- A conventional lab coat, closed with long sleeves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/9/9c/Lab_coat.png"></td></tr><br />
<tr><td><p class="texte">- Closed shoes</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/8/82/Shoes.png"></td></tr><br />
<tr><td><p class="texte">- Gloves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/3/37/Gloves.png"></td></tr><br />
<tr><td><p class="texte">- Glasses if needed (UV exposure, hot water or chemicals manipulation.) </p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/b/b4/Glasses.png"></td></tr><br />
</table><br />
<br />
<p class="title3">Basic rules in a lab</p><br />
<p class="texte">We have to apply the basic safety principles into a laboratory room:<br><br />
- It is forbidden to smoke in all rooms.<br><br />
- It is forbidden to drink and eat in the laboratory rooms.<br><br />
- It is compulsory to wear a closed lab coat in cotton.<br><br />
- It is compulsory to wear closed shoes.<br><br />
- Long hair must be tied back.<br><br />
- Oral pipetting of any substance is prohibited in any laboratory.<br><br />
<br><br />
There are also others precautions when working with biological organisms:<br><br />
- We need to wash our hands regularly.<br><br />
- It is compulsory to wear gloves except with the use of an electric burner.<br><br />
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.<br><br />
<br><br />
Different apparatus are used to work a sterile area.</p><br />
<br />
<p class="title3">Waste</p><br />
<p class="texte">Different trash containers are available in the lab:<br><br />
- One for biological waste (yellow). This waste will be autoclaved before being thrown out.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/f7/Yellow_trash.JPG"><br><br><br />
- One for common waste (green or orange).<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/c/c7/Black_garbage.png"><br><br><br />
- Special waste for chemicals.<br />
<br />
<p class="title3">Devices and Material</p><br />
<p class="texte">We use a lot of different devices, and each one involves a particular risk. Here we described how we use this material safely to reduce risks:</p><br />
<p class="title4">Chemical storage</p><br />
<p class="texte">We have three cupboards dedicated to the different kind of chemical products we use:<br><br />
- Flammable<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/35/Flammable.png"><br><br><br />
- Acids<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/36/Acids.png"><br><br><br />
- Bases<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/74/Bases.png"><br><br><br />
Those cupboards are key-closed.</p><br />
<br />
<p class="title4">Ethidium Bromide</p><br />
<p class="texte">We have a dark room dedicated to the use of EtBr and UV. This room is key-closed and everyone entering into the room must wear gloves, glasses and lab coat. Everything which is in direct contact with something in this room has to stay here.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/75/EtBr_room.JPG"><br><br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/4/45/EtBr_door.JPG"><br><br><br />
Two specific trash cans are dedicated to the gloves or paper and the agarose gels contaminated.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/fe/EtBr_garbage.JPG"><br><br></p><br />
<br />
<p class="title4">Biological safety cabinet</p><br />
<p class="texte">We use a Biological safety cabinet (FASTER – Ultrasafe) to manipulate into a sterile area and thus avoid external contamination by unwanted microorganisms. We clean the bench of the BSC with ethanol before and after each manipulation. We also clean the BSC completely every two weeks. Because we work with fungi, we have to be very careful with the cleanliness of the BSC.</p><br />
<br />
<p class="title4">Electric burner</p><br />
<p class="texte">We also use electric burners to manipulate into a sterile area. The burning and fire risks are high but weaker than with a Bunsen Burner. We are very cautious when we use electric burners, and we use in preference BSC if it is possible. We do not have to wear gloves and we have to check that the electric burner is turned off after the manipulation.</p><br />
<br />
<p class="title4">Chemical hood</p><br />
<p class="texte">We use chemical hood in case we have to manipulate dangerous volatile chemical compound.</p><br />
<br />
<p class="title4">Water-bathes</p><br />
<p class="texte">The water-bathes are used extensively (transformation, digestion, etc.). However, they can be dangerous because of the exposition of boiling or hot water. We use special gloves for protecting us from heat, steam and projections. We verify that we have turned off the water-bathes at the end of the day.</p><br />
<br />
<br />
<br />
<p class="title2">Safety of our project</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"Precaution is better than cure"</p><br />
<p class="texte">Several risks exist when working with microorganisms and manipulating genes. We can identify different classes of risk of our project now: <br><br />
- risks to the safety and health of team members, or other people working in the lab,<br><br />
- risks to the safety and health of the general public,<br><br />
- risks to the environment,<br><br />
- risks to security through malicious use by individuals, groups, or countries.<br><br />
<br><br />
There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).<br><br />
<br />
<p class="title3">Risks to the safety and health of team members, or other people working in the lab</p><br />
<p class="texte">We work with the <I>B. subtilis </I> and <i>E. coli</i> chassis. These organisms are non-pathogenic. Moreover the used parts are also non-pathogenic (the bio-fungicides produced are harmless for humans).<br><br />
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.<br><br />
On the other hand the main risk for humans is chemical: we use Ethidium Bromide, hydrochloric acid, sodium hydroxide, solutions from a Miniprep kit, ethanol and bleach.<br><br />
<br><br />
However, we have a black room dedicated to the manipulation of Ethidium Bromide. Everything that touches something in this room cannot get out of the room. The waste has a special treatment.<br><br />
For the other chemical solutions, we always use gloves and glasses, and if necessary a hood.<br><br />
Furthermore, there are other risks such as UVs when we analyze an agarose gel but also water-bath and electrical/fire risks.<br><br />
For protecting us from the UVs, we use adapted glasses.<br><br />
At the end of every day, the last persons to leave check that everything is ok in the lab (no water-bath stayed on, everything disconnected).</p><br />
<br />
<p class="title3">Risks to the safety and health of the general public</p><br />
<p class="texte">If biological materials escape from our lab, the risks regarding safety and health of the general public is low because the manipulated parts are harmless. The only risk is the contact with DNA or RNA from a bacterial organism.<br><br />
Moreover, we have to be cautious with the use of antibiotics and we must not wash them down the drain.</p><br />
<br />
<p class="title3">Risks to the environment</p><br />
<p class="texte">If biological materials escape from our lab, GMOs can spread in the environment and gene transfer can occur. Moreover, the aim of our bacterial system (called SubtiTree) is to kill a fungus (Ceratocyctis platani). The risk to the environment is high because our system could perturb ecological niche and unbalance the environment of some plants. All our waste is autoclaved to minimize the risk for the environment.<br><br />
We use three fungicides.<br><br />
The first one, D4E1, is a synthetic peptide.<br><br />
The second is EcAMP-1 (BBa_K1162001), an AntiMicrobial Peptide from banyard grass added to the Registry last year by the team Utah State.<br><br />
The last one is GAFP-1, the Gastrodia AntiFungal Protein.<br><br />
These fungicides are not authorized yet by the European Union because they are not commercialized.</p><br />
<br />
<p class="title3">Risks to security through malicious mis-use by individuals, groups, or countries</p><br />
<p class="texte">If someone steals the biological material it could be dangerous for the environment because our bacterium will produce three bio-fungicides. This could lead to the disturbing of some ecosystems. <br><br />
However, the fungicides are natural and one of them is used in agriculture. With these considerations, we can assume that the risk is not very high for the chosen parts.</p><br />
<br />
<br />
<p class="title3">How to reduce these risks?</p><br />
<p class="texte">We take care of our waste and our biological material.<br><br />
We have chosen only non-pathogenic species to work on: <i>E. coli</i> and <i>B. subtilis</i>.<br><br />
We use non-pathogenic fungus to test our SubtiTree system: <i>Aspergillus brasiliensis</i>, <i>Chaetomium globosum</i>, <i>Trichoderma reesei</i> and <i>Aspergillus nidulans</i>.<br><br />
Indeed the targeted fungus, Ceratocystis platani, is pathogenic. Therefore we avoid working with it.</p><br />
<br />
<p class="title3">Risks in the Future</p><br />
<p class="texte">The system designed could be applied to many fungi. The fungicides used in our project are biological and specifically kill the fungi, and no other eukaryotic cells. <br><br />
However, a malicious person could replace these fungicides by others which are more dangerous. Indeed our problem is that there is no regulation of the production of fungicides.</p><br />
<br />
<p class="title3">How to reduce these Future risks?</p><br />
<p class="texte">We have thought about several ways to minimize risks: <br><br />
- we can build an auxotrophic strain of <I>B. subtilis</I>. The auxotrophy will be for proline, which is readily present in the plant's sap.<br><br />
- we can build a non-sporulating strain of<I>B. subtilis</I>. Sporulation is a bacterial mechanism used to survive through winter. Thus, our bacterial system sould not stay in the tree after the first year cycle.<br><br />
- we can build a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and any other wild-type bacteria.</p><br />
<br />
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<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Safety</p> <br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">iGEM Safety</a></li><br />
<li><a href="#select2">Chassis Organism</a></li><br />
<li><a href="#select3">New and/or modified coding region</a></li><br />
<li><a href="#select4">Team safety</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Safety is not just a slogan, it's a way of life"<br />
</p><br />
<br />
<br><br />
<!--PARTIE iGEM SAFETY--><br />
<p class="title1" id="select1">iGEM Safety</p><br />
<p class ="texte">Our safety form was approved in September 2014 by the iGEM team. We filled it with the help of Nathalie Doubrovine, safety officer at the LISBP.<br />
</p><br />
<br />
<!--TABLE CHASSIS ORGANISM--><br />
<p class="title1" id="select2">Chassis organisms</p><br />
<br />
<table style="border-width:1px; border-color:#ccc;width:100%;background-color:#ccc;"<br />
><br />
<tbody style="background-color:white;"><br />
<br />
<!--1ere ligne--><br />
<tr><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 30px;">Species name (including strain)</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px;">Risk group</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px;">Risk Group Source</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; padding-top: 42px; font-size: 17px; ">Disease risk for humans ?</td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td><i><p class ="texte" style="padding-top: 10px; text-align:center;">Escherichia coli MC1061</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-7140.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Bacillus subtilis 168</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-23778.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Aspergillus brasiliensis 246.65 CBS</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-63263.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Chaetomium globosum 148.51 CBS</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-1962.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Trichoderma reesei CBS 383.78</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-768.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Aspergillus nidulans CBS 124.59</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-820.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
<br><br><br />
<br />
<br />
<!--TABLE NEW AND/OR MODIFIED CODING REGION--><br />
<p class="title1" id="select3">New and/or modified coding region</p><br />
<br />
<table style="border-width:1px; border-color:#ccc;width:100%;background-color:#ccc;"<br />
><br />
<tbody style="background-color:white;"><br />
<br />
<!--1ere ligne--><br />
<tr><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 22px 10px; font-size: 15px;">Part number/name</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">Natural function of part</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">How did you acquire it?</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">How will you use it?</p></td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">N-acetylatedGlucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS - Antifungal GAFP-1</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + N-acetyl-glucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + Chitin Binding Protein for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allow the bacterium to fix the fungus</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364006">BBa_K1364006</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Double expression cassette</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach and bind to the fungus</p></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS + Antifungal GAFP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</p></td><br />
<td><p class ="texte">P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub>-SpoVG + EcAMP-1</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub>-spoVG + EcAMP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--14eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RFP</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fluorescent protein. By this way we can evaluate promotor P<sub>veg</sub> strength.</p></td><br />
</tr><br />
<!--16eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>lepA</sub> + RFP</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fluorescent protein. By this way we can evaluate promotor P<sub>lepA</sub> strength.</p></td><br />
</tr><br />
<!--17eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Promotor P<sub>lepA</sub> + RBS spoVG</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part provides us a high level of translation.</p></td><br />
</tr><br />
<!--18eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RBS + Antifungal EcAMP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus.</p></td><br />
</tr><br />
<!--19eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Integrative plasmid for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part is an integrative vector.</p></td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
<br><br />
<br />
<p class="title1" id="select4">Team safety</p><br />
<p class="title2">Safety in INSA de Toulouse</p><br />
<p class="texte">INSA de Toulouse is a public school for engineers. The biosafety guidelines are not specific to our institution; the French regulations are applied.<br><br />
The regulation about the workers' prevention against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994) includes microorganisms, cell cultures and human endoparasites which may cause infections, allergies or toxicity. <br><br />
This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5.)<br><br />
The <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000465273&dateTexte=&categorieLien=id">Decree of the 16th July 2007</a> describes the technical preventive measuresto set up in research laboratories (including containment), education, analysis, anatomy and surgical pathology, autopsy rooms, and industrial and agricultural facilities where workers are likely to be exposed to biological pathogens.<br><br />
The rules of health, safety, and preventive medicine applied in public services in France (and thus in all public facilities working in scientific and technological domains) are set out in the <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=LEGITEXT000006063791">Decree No. 82-453</a>. This decree refers to the Labour Code, Public Health Code and Environmental Code.<br><br />
The <a href="http://legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000024584737&categorieLien=id">Decree No. 2011-1177</a> is related to the use of genetically modified organisms.<br><br />
<br><br />
There is no one in charge of the biological safety at the INSA de Toulouse. However there is an organization which includes one prevention advisor and several prevention assistants in every structure. As far as the LISBP (our structure) is concerned, Nathalie Doubrovine is the prevention assistant.<br><br />
We have already discussed our project with her. She has given us the safety training and advices about how to respond to an imminent danger.<br><br />
She raised the problem of GMO and the related policy. Indeed she told us there is a special procedure for the destruction of GMO: we cannot wash this kind of organism down the drain.<br><br />
We were also informed about the potential chemical and microbiological risks thanks to her.<br><br />
<br><br />
The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedure of the LISBP.<br><br />
The legislation requires employers to inform "all new employees" of the risks that they may encounter as well as ways to protect themselves from those risks. Therefore any newcomer must pass a practical and appropriate training in hygiene and safety to ensure its own safety and the one of his colleagues.<br><br />
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).<br><br />
The training is divided into two parts. The first one is a training concerning general risk prevention into research laboratories. This one is made individually thanks to the software NEO and with the explanations of the prevention assistant. The second one is a training about the techniques used during the occupation.<br><br />
<br><br />
All the team members have received this safety training. It lasts 1h30. It concerns fire risks, biological and chemical risks and good laboratory practices. We have made this training individually with a software designed by the CNRS and INSERM named NEO. This training ends with a questionnaire to check our knowledge. This questionnaire reminds us the basic safety rules in a molecular biology lab and gives us new knowledge about safety.<br><br />
We have also made a training to know how to sort out biological waste and how they are treated. We also learned that we cannot use autoclave by ourselves because a specific training is needed. We did not do this specific training.<br><br />
</p><br />
<br />
<p class="title2">Safety in the lab</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"If you don't think it's safe, it probably isn't"</p><br />
<p class="texte">We have organized our workspace. There is the relaxing room where no microbial material should enter. In this room, we can eat and drink (a fridge, a kettle and a coffeemaker are available) but it is also the room where we have our meetings and where we can work on our computers. There is the lab, where we have to wear protective equipment and respect basic rules.</p><br />
<br />
<p class="title3">Personal protective equipment</p><br />
<p class="texte">As soon as we manipulate in the lab, we have to wear the following personal protective equipment:</p><br />
<table><br />
<tr><td><p class="texte">- A conventional lab coat, closed with long sleeves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/9/9c/Lab_coat.png"></td></tr><br />
<tr><td><p class="texte">- Closed shoes</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/8/82/Shoes.png"></td></tr><br />
<tr><td><p class="texte">- Gloves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/3/37/Gloves.png"></td></tr><br />
<tr><td><p class="texte">- Glasses if needed (UV exposure, hot water or chemicals manipulation.) </p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/b/b4/Glasses.png"></td></tr><br />
</table><br />
<br />
<p class="title3">Basic rules in a lab</p><br />
<p class="texte">We have to apply the basic safety principles into a laboratory room:<br><br />
- It is forbidden to smoke in all the rooms.<br><br />
- It is forbidden to drink and eat in the laboratory rooms.<br><br />
- It is compulsory to wear a closed lab coat in cotton.<br><br />
- It is compulsory to wear closed shoes.<br><br />
- Long hair must be tied back.<br><br />
- Oral pipetting of any substance is prohibited in any laboratory.<br><br />
<br><br />
There is also others precaution when working with biological organisms:<br><br />
- We need to wash our hands regularly.<br><br />
- It is compulsory to wear gloves except with the use of an electric burner.<br><br />
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.<br><br />
<br><br />
Indeed, different machine are used to keep a sterile area.</p><br />
<br />
<p class="title3">Waste</p><br />
<p class="texte">Different trash containers are available in the lab:<br><br />
- One for biological waste (yellow). This waste will be autoclaved.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/f7/Yellow_trash.JPG"><br><br><br />
- One for common waste (green or orange).<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/c/c7/Black_garbage.png"><br><br><br />
- Special waste for chemicals.<br />
<br />
<p class="title3">Devices and Material</p><br />
<p class="texte">We use a lot of different devices, and each one involves a particular risk. Here we described how we use this material safely to reduce risks:</p><br />
<p class="title4">Chemical storage</p><br />
<p class="texte">We have three cupboards dedicated to the different kind of chemical products we use:<br><br />
- Flammable<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/35/Flammable.png"><br><br><br />
- Acids<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/36/Acids.png"><br><br><br />
- Bases<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/74/Bases.png"><br><br><br />
Those cupboards are key-closed.</p><br />
<br />
<p class="title4">Ethidium Bromide</p><br />
<p class="texte">We have a dark room dedicated to the use of EtBr and UV. This room is key-closed and everyone entering into the room must wear gloves, glasses and lab coat. Everything which is in direct contact with something in this room has to stay here.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/75/EtBr_room.JPG"><br><br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/4/45/EtBr_door.JPG"><br><br><br />
Two specific trash cans are dedicated to the gloves or paper and the agarose gels contaminated.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/fe/EtBr_garbage.JPG"><br><br></p><br />
<br />
<p class="title4">Biological safety cabinet</p><br />
<p class="texte">We use a Biological safety cabinet (FASTER – Ultrasafe) to manipulate into a sterile area and thus avoid external contamination by unwanted microorganisms. We clean the bench of the BSC with ethanol before and after each manipulation. We also clean the BSC completely every two weeks. Because we work with fungi, we have to be very careful with the cleanliness of the BSC.</p><br />
<br />
<p class="title4">Electric burner</p><br />
<p class="texte">We also use electric burners to manipulate into a sterile area. The burning and fire risks are high but weaker than with a Bunsen Burner. We are very cautious when we use electric burners, and we use in preference BSC if it is possible. We do not have to wear gloves et we have to check that the electric burner is turned off after the manipulation.</p><br />
<br />
<p class="title4">Chemical hood</p><br />
<p class="texte">We use chemical hood in case we have to manipulate dangerous volatile chemical compound.</p><br />
<br />
<p class="title4">Water-bathes</p><br />
<p class="texte">The water-bathes are used extensively (transformation, digestion, etc.). However, they can be dangerous because of the exposition of boiling or hot water. We use special gloves for protecting us from heat, steam and projections. We take care of turning off the water-bathes at the end of the day.</p><br />
<br />
<br />
<br />
<p class="title2">Safety of our project</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"Precaution is better than cure"</p><br />
<p class="texte">Several risks exist when working with microorganisms and manipulating genes. We can identify different classes of risk of our project now: <br><br />
- risks to the safety and health of team members, or other people working in the lab,<br><br />
- risks to the safety and health of the general public,<br><br />
- risks to the environment,<br><br />
- risks to security through malicious mis-use by individuals, groups, or countries.<br><br />
<br><br />
There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).<br><br />
<br />
<p class="title3">Risks to the safety and health of team members, or other people working in the lab</p><br />
<p class="texte">We work with the <I>B. subtilis </I> and <i>E. coli</i> chassis. These organisms are non-pathogenic. Moreover the used parts are also non-pathogenic (the bio-fungicides produced are harmless for humans).<br><br />
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.<br><br />
On the other hand the main risk for humans is chemical: we use Ethidium Bromide, hydrochloric acid, soda, solutions from a Miniprep kit, ethanol and bleach.<br><br />
<br><br />
However, we have a black room dedicated to the manipulation of Ethidium Bromide. Everything that touches something in this room cannot get out of the room. The waste has a special treatment.<br><br />
For the other chemical solutions, we always use gloves and glasses, and if necessary a hood.<br><br />
Furthermore, there are other risks such as UVs when we analyze an agarose gel but also water-bath and electrical/fire risks.<br><br />
For protecting us from the UVs, we use adapted glasses.<br><br />
At the end of every day, the last persons to leave check that everything is ok in the lab (no water-bath stayed on, everything disconnected).</p><br />
<br />
<p class="title3">Risks to the safety and health of the general public</p><br />
<p class="texte">If biological materials escape from our lab, the risks regarding safety and health of the general public is low because the manipulated parts are harmless. The only risk is the contact with DNA or RNA from a bacterial organism.<br><br />
Moreover, we have to be cautious with the use of antibiotics and we must not wash them down the drain.</p><br />
<br />
<p class="title3">Risks to the environment</p><br />
<p class="texte">If biological materials escape from our lab, GMOs can spread in the environment and gene transfer can occur. Moreover, the aim of our bacterial system (called SubtiTree) is to kill a fungus (Ceratocyctis platani). The risk to the environment is high because our system could perturb ecological niche and unbalance the environment of some plants. All our waste is autoclaved to minimize the risk for the environment.<br><br />
We use three fungicides.<br><br />
The first one, D4E1, is a synthetic peptide.<br><br />
The second is EcAMP-1 (BBa_K1162001), an AntiMicrobial Peptide from banyard grass added to the Registry last year by the team Utah State.<br><br />
The last one is GAFP-1, the Gastrodia AntiFungal Protein.<br><br />
These fungicides are not authorized yet by the European Union because they are not commercialized.</p><br />
<br />
<p class="title3">Risks to security through malicious mis-use by individuals, groups, or countries</p><br />
<p class="texte">If someone steals the biological material it could be dangerous for the environment because our bacterium will produce three bio-fungicides. This could lead to the disturbing of some ecosystems. <br><br />
However, the fungicides are natural and one of them is used in agriculture. With these considerations, we can assume that the risk is not very high for the chosen parts.</p><br />
<br />
<br />
<p class="title3">How to reduce these risks?</p><br />
<p class="texte">We take care of our waste and our biological material.<br><br />
We have chosen only non-pathogenic species to work on: <i>E. coli</i> and <i>B. subtilis</i>.<br><br />
We use non-pathogenic fungus to test our SubtiTree system: <i>Aspergillus brasiliensis</i>, <i>Chaetomium globosum</i>, <i>Trichoderma reesei</i> and <i>Aspergillus nidulans</i>.<br><br />
Indeed the targeted fungus, Ceratocystis platani, is pathogenic. Therefore we avoid working with it.</p><br />
<br />
<p class="title3">Risks in the Future</p><br />
<p class="texte">The system designed could be applied to many fungi. The fungicides used in our project are biological and specifically kill the fungi, and no other eukaryotic cells. <br><br />
However, a malicious person could replace these fungicides by others which are more dangerous. Indeed our problem is that there is no regulation of the production of fungicides.</p><br />
<br />
<p class="title3">How to reduce these Future risks?</p><br />
<p class="texte">We have thought about several ways to minimize risks: <br><br />
- we choose an auxotrophic strain of <I>B. subtilis</I>. The auxotrophy is for the glutamic acid, which is very present in plant's sap.<br><br />
- we choose a non-spore-forming strain of<I>B. subtilis</I>. The sporulation is the endophyte bacteria mechanism used to survive through winter. Thus, our bacterial system will be time limited.<br><br />
- we want to use a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and another wild-type bacteria.</p><br />
<br />
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<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Safety</p> <br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">iGEM Safety</a></li><br />
<li><a href="#select2">Chassis Organism</a></li><br />
<li><a href="#select3">New and/or modified coding region</a></li><br />
<li><a href="#select4">Team safety</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Safety is not just a slogan, it's a way of life"<br />
</p><br />
<br />
<br><br />
<!--PARTIE iGEM SAFETY--><br />
<p class="title1" id="select1">iGEM Safety</p><br />
<p class ="texte">Our safety form was approved in September 2014 by the iGEM team. We filled it with the help of Nathalie Doubrovine, safety officer at the LISBP.<br />
</p><br />
<br />
<!--TABLE CHASSIS ORGANISM--><br />
<p class="title1" id="select2">Chassis organisms</p><br />
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<table style="border-width:1px; border-color:#ccc;width:100%;background-color:#ccc;"<br />
><br />
<tbody style="background-color:white;"><br />
<br />
<!--1ere ligne--><br />
<tr><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 30px;">Species name (including strain)</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px;">Risk group</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px;">Risk Group Source</td><br />
<td style=" background-color:#22780F"><p class ="texte" style="color:white; text-align:center; padding-top: 42px; font-size: 17px; ">Disease risk for humans ?</td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td><i><p class ="texte" style="padding-top: 10px; text-align:center;">Escherichia coli MC1061</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-7140.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Bacillus subtilis 168</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-23778.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Aspergillus brasiliensis 246.65 CBS</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-63263.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Chaetomium globosum 148.51 CBS</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-1962.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Trichoderma reesei CBS 383.78</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-768.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="padding-top: 10px; text-align:center;"><i>Aspergillus nidulans CBS 124.59</i></p></td><br />
<td><p class ="texte" style="text-align:center">1</p></td><br />
<td><p class ="texte" style="text-align:center"><a href="http://www.dsmz.de/catalogues/details/culture/DSM-820.html">DSMZ</p></a><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)</p></td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
<br><br><br />
<br />
<br />
<!--TABLE NEW AND/OR MODIFIED CODING REGION--><br />
<p class="title1" id="select3">New and/or modified coding region</p><br />
<br />
<table style="border-width:1px; border-color:#ccc;width:100%;background-color:#ccc;"<br />
><br />
<tbody style="background-color:white;"><br />
<br />
<!--1ere ligne--><br />
<tr><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 22px 10px; font-size: 15px;">Part number/name</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">Natural function of part</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">How did you acquire it?</p></td><br />
<td style="background-color:#22780F"><p class ="texte" style="color:white; text-align:center; margin: 0 10px; font-size: 15px;">How will you use it?</p></td><br />
</tr><br />
<!--2eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">N-acetylatedGlucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a synthesis company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS - Antifungal GAFP-1</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a synthesis company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacteria is able to kill the fungus.</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We ordered this gene from a synthesis company (Eurofins)</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacteria is able to kill the fungus.</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach the fungus</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + Chitin Binding Protein for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allow the bacterium to fix the fungus</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364006">BBa_K1364006</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Double expression cassette</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part allows the bacterium to reach and bind to the fungus</p></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">RBS + Antifungal GAFP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</p></td><br />
<td><p class ="texte">P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub>-SpoVG + EcAMP-1</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub>-spoVG + EcAMP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus</p></td><br />
</tr><br />
<!--14eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus.</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RFP</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fluorescent protein. By this way we can evaluate promotor P<sub>veg</sub> strength.</p></td><br />
</tr><br />
<!--16eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>lepA</sub> + RFP</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fluorescent protein. By this way we can evaluate promotor P<sub>lepA</sub> strength.</p></td><br />
</tr><br />
<!--17eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Promotor P<sub>lepA</sub> + RBS spoVG</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part provides us a high level of translation.</p></td><br />
</tr><br />
<!--18eme ligne--><br />
<tr style=" background-color:#f8f8f8"><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RBS + Antifungal EcAMP-1 + Double terminator</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide. By this way, the bacterium is able to kill the fungus.</p></td><br />
</tr><br />
<!--19eme ligne--><br />
<tr><br />
<td><p class ="texte" style="text-align:center;"><a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">Integrative plasmid for <i>Bacillus subtilis</i></p></td><br />
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td><br />
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part is an integrative vector.</p></td><br />
</tr><br />
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</tbody><br />
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<p class="title1" id="select4">Team safety</p><br />
<p class="title2">Safety in INSA de Toulouse</p><br />
<p class="texte">INSA de Toulouse is a public school for engineers. The biosafety guidelines are not specific to our institution; the French regulations are applied.<br><br />
The regulation about the workers' prevention against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994) includes microorganisms, cell cultures and human endoparasites which may cause infections, allergies or toxicity. <br><br />
This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5.)<br><br />
The <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000465273&dateTexte=&categorieLien=id">Decree of the 16th July 2007</a> describes the technical preventive measuresto set up in research laboratories (including containment), education, analysis, anatomy and surgical pathology, autopsy rooms, and industrial and agricultural facilities where workers are likely to be exposed to biological pathogens.<br><br />
The rules of health, safety, and preventive medicine applied in public services in France (and thus in all public facilities working in scientific and technological domains) are set out in the <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=LEGITEXT000006063791">Decree No. 82-453</a>. This decree refers to the Labour Code, Public Health Code and Environmental Code.<br><br />
The <a href="http://legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000024584737&categorieLien=id">Decree No. 2011-1177</a> is related to the use of genetically modified organisms.<br><br />
<br><br />
There is no one in charge of the biological safety at the INSA de Toulouse. However there is an organization which includes one prevention advisor and several prevention assistants in every structure. As far as the LISBP (our structure) is concerned, Nathalie Doubrovine is the prevention assistant.<br><br />
We have already discussed our project with her. She has given us the safety training and advices about how to respond to an imminent danger.<br><br />
She raised the problem of GMO and the related policy. Indeed she told us there is a special procedure for the destruction of GMO: we cannot wash this kind of organism down the drain.<br><br />
We were also informed about the potential chemical and microbiological risks thanks to her.<br><br />
<br><br />
The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedure of the LISBP.<br><br />
The legislation requires employers to inform "all new employees" of the risks that they may encounter as well as ways to protect themselves from those risks. Therefore any newcomer must pass a practical and appropriate training in hygiene and safety to ensure its own safety and the one of his colleagues.<br><br />
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).<br><br />
The training is divided into two parts. The first one is a training concerning general risk prevention into research laboratories. This one is made individually thanks to the software NEO and with the explanations of the prevention assistant. The second one is a training about the techniques used during the occupation.<br><br />
<br><br />
All the team members have received this safety training. It lasts 1h30. It concerns fire risks, biological and chemical risks and good laboratory practices. We have made this training individually with a software designed by the CNRS and INSERM named NEO. This training ends with a questionnaire to check our knowledge. This questionnaire reminds us the basic safety rules in a molecular biology lab and gives us new knowledge about safety.<br><br />
We have also made a training to know how to sort out biological waste and how they are treated. We also learned that we cannot use autoclave by ourselves because a specific training is needed. We did not do this specific training.<br><br />
</p><br />
<br />
<p class="title2">Safety in the lab</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"If you don't think it's safe, it probably isn't"</p><br />
<p class="texte">We have organized our workspace. There is the relaxing room where no microbial material should enter. In this room, we can eat and drink (a fridge, a kettle and a coffeemaker are available) but it is also the room where we have our meetings and where we can work on our computers. There is the lab, where we have to wear protective equipment and respect basic rules.</p><br />
<br />
<p class="title3">Personal protective equipment</p><br />
<p class="texte">As soon as we manipulate in the lab, we have to wear the following personal protective equipment:</p><br />
<table><br />
<tr><td><p class="texte">- A conventional lab coat, closed with long sleeves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/9/9c/Lab_coat.png"></td></tr><br />
<tr><td><p class="texte">- Closed shoes</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/8/82/Shoes.png"></td></tr><br />
<tr><td><p class="texte">- Gloves</p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/3/37/Gloves.png"></td></tr><br />
<tr><td><p class="texte">- Glasses if needed (UV exposure, hot water or chemicals manipulation.) </p></td><br />
<td><img width="80px" src="https://static.igem.org/mediawiki/2014/b/b4/Glasses.png"></td></tr><br />
</table><br />
<br />
<p class="title3">Basic rules in a lab</p><br />
<p class="texte">We have to apply the basic safety principles into a laboratory room:<br><br />
- It is forbidden to smoke in all the rooms.<br><br />
- It is forbidden to drink and eat in the laboratory rooms.<br><br />
- It is compulsory to wear a closed lab coat in cotton.<br><br />
- It is compulsory to wear closed shoes.<br><br />
- Long hair must be tied back.<br><br />
- Oral pipetting of any substance is prohibited in any laboratory.<br><br />
<br><br />
There is also others precaution when working with biological organisms:<br><br />
- We need to wash our hands regularly.<br><br />
- It is compulsory to wear gloves except with the use of an electric burner.<br><br />
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.<br><br />
<br><br />
Indeed, different machine are used to keep a sterile area.</p><br />
<br />
<p class="title3">Waste</p><br />
<p class="texte">Different trash containers are available in the lab:<br><br />
- One for biological waste (yellow). This waste will be autoclaved.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/f7/Yellow_trash.JPG"><br><br><br />
- One for common waste (green or orange).<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/c/c7/Black_garbage.png"><br><br><br />
- Special waste for chemicals.<br />
<br />
<p class="title3">Devices and Material</p><br />
<p class="texte">We use a lot of different devices, and each one involves a particular risk. Here we described how we use this material safely to reduce risks:</p><br />
<p class="title4">Chemical storage</p><br />
<p class="texte">We have three cupboards dedicated to the different kind of chemical products we use:<br><br />
- Flammable<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/35/Flammable.png"><br><br><br />
- Acids<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/3/36/Acids.png"><br><br><br />
- Bases<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/74/Bases.png"><br><br><br />
Those cupboards are key-closed.</p><br />
<br />
<p class="title4">Ethidium Bromide</p><br />
<p class="texte">We have a dark room dedicated to the use of EtBr and UV. This room is key-closed and everyone entering into the room must wear gloves, glasses and lab coat. Everything which is in direct contact with something in this room has to stay here.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/7/75/EtBr_room.JPG"><br><br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/4/45/EtBr_door.JPG"><br><br><br />
Two specific trash cans are dedicated to the gloves or paper and the agarose gels contaminated.<br><br />
<img width="150px" src="https://static.igem.org/mediawiki/2014/f/fe/EtBr_garbage.JPG"><br><br></p><br />
<br />
<p class="title4">Biological safety cabinet</p><br />
<p class="texte">We use a Biological safety cabinet (FASTER – Ultrasafe) to manipulate into a sterile area and thus avoid external contamination by unwanted microorganisms. We clean the bench of the BSC with ethanol before and after each manipulation. We also clean the BSC completely every two weeks. Because we work with fungi, we have to be very careful with the cleanliness of the BSC.</p><br />
<br />
<p class="title4">Electric burner</p><br />
<p class="texte">We also use electric burners to manipulate into a sterile area. The burning and fire risks are high but weaker than with a Bunsen Burner. We are very cautious when we use electric burners, and we use in preference BSC if it is possible. We do not have to wear gloves et we have to check that the electric burner is turned off after the manipulation.</p><br />
<br />
<p class="title4">Chemical hood</p><br />
<p class="texte">We use chemical hood in case we have to manipulate dangerous volatile chemical compound.</p><br />
<br />
<p class="title4">Water-bathes</p><br />
<p class="texte">The water-bathes are used extensively (transformation, digestion, etc.). However, they can be dangerous because of the exposition of boiling or hot water. We use special gloves for protecting us from heat, steam and projections. We take care of turning off the water-bathes at the end of the day.</p><br />
<br />
<br />
<br />
<p class="title2">Safety of our project</p><br />
<!--CITATION--><br />
<p class="citation"><br />
"Precaution is better than cure"</p><br />
<p class="texte">Several risks exist when working with microorganisms and manipulating genes. We can identify different classes of risk of our project now: <br><br />
- risks to the safety and health of team members, or other people working in the lab,<br><br />
- risks to the safety and health of the general public,<br><br />
- risks to the environment,<br><br />
- risks to security through malicious mis-use by individuals, groups, or countries.<br><br />
<br><br />
There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).<br><br />
<br />
<p class="title3">Risks to the safety and health of team members, or other people working in the lab</p><br />
<p class="texte">We work with the <I>B. subtilis </I> and <i>E. coli</i> chassis. These organisms are non-pathogenic. Moreover the used parts are also non-pathogenic (the bio-fungicides produced are harmless for humans).<br><br />
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.<br><br />
On the other hand the main risk for humans is chemical: we use Ethidium Bromide, hydrochloric acid, soda, solutions from a Miniprep kit, ethanol and bleach.<br><br />
<br><br />
However, we have a black room dedicated to the manipulation of Ethidium Bromide. Everything that touches something in this room cannot get out of the room. The waste has a special treatment.<br><br />
For the other chemical solutions, we always use gloves and glasses, and if necessary a hood.<br><br />
Furthermore, there are other risks such as UVs when we analyze an agarose gel but also water-bath and electrical/fire risks.<br><br />
For protecting us from the UVs, we use adapted glasses.<br><br />
At the end of every day, the last persons to leave check that everything is ok in the lab (no water-bath stayed on, everything disconnected).</p><br />
<br />
<p class="title3">Risks to the safety and health of the general public</p><br />
<p class="texte">If biological materials escape from our lab, the risks regarding safety and health of the general public is low because the manipulated parts are harmless. The only risk is the contact with DNA or RNA from a bacterial organism.<br><br />
Moreover, we have to be cautious with the use of antibiotics and we must not wash them down the drain.</p><br />
<br />
<p class="title3">Risks to the environment</p><br />
<p class="texte">If biological materials escape from our lab, GMOs can spread in the environment and gene transfer can occur. Moreover, the aim of our bacterial system (called SubtiTree) is to kill a fungus (Ceratocyctis platani). The risk to the environment is high because our system could perturb ecological niche and unbalance the environment of some plants. All our waste is autoclaved to minimize the risk for the environment.<br><br />
We use three fungicides.<br><br />
The first one, D4E1, is a synthetic peptide.<br><br />
The second is EcAMP-1 (BBa_K1162001), an AntiMicrobial Peptide from banyard grass added to the Registry last year by the team Utah State.<br><br />
The last one is GAFP-1, the Gastrodia AntiFungal Protein.<br><br />
These fungicides are not authorized yet by the European Union because they are not commercialized.</p><br />
<br />
<p class="title3">Risks to security through malicious mis-use by individuals, groups, or countries</p><br />
<p class="texte">If someone steals the biological material it could be dangerous for the environment because our bacterium will produce three bio-fungicides. This could lead to the disturbing of some ecosystems. <br><br />
However, the fungicides are natural and one of them is used in agriculture. With these considerations, we can assume that the risk is not very high for the chosen parts.</p><br />
<br />
<br />
<p class="title3">How to reduce these risks?</p><br />
<p class="texte">We take care of our waste and our biological material.<br><br />
We have chosen only non-pathogenic species to work on: <i>E. coli</i> and <i>B. subtilis</i>.<br><br />
We use non-pathogenic fungus to test our SubtiTree system: <i>Aspergillus brasiliensis</i>, <i>Chaetomium globosum</i>, <i>Trichoderma reesei</i> and <i>Aspergillus nidulans</i>.<br><br />
Indeed the targeted fungus, Ceratocystis platani, is pathogenic. Therefore we avoid working with it.</p><br />
<br />
<p class="title3">Risks in the Future</p><br />
<p class="texte">The system designed could be applied to many fungi. The fungicides used in our project are biological and specifically kill the fungi, and no other eukaryotic cells. <br><br />
However, a malicious person could replace these fungicides by others which are more dangerous. Indeed our problem is that there is no regulation of the production of fungicides.</p><br />
<br />
<p class="title3">How to reduce these Future risks?</p><br />
<p class="texte">We have thought about several ways to minimize risks: <br><br />
- we choose an auxotrophic strain of <I>B. subtilis</I>. The auxotrophy is for the glutamic acid, which is very present in plant's sap.<br><br />
- we choose a non-spore-forming strain of<I>B. subtilis</I>. The sporulation is the endophyte bacteria mechanism used to survive through winter. Thus, our bacterial system will be time limited.<br><br />
- we want to use a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and another wild-type bacteria.</p><br />
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