Team:Toulouse/Safety

From 2014.igem.org

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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<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>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
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<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>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
+
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
-
<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>
+
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<td><p class ="texte">P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p></td>
<td><p class ="texte">P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
+
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
+
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
-
<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>
+
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
-
<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>
+
<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RFP</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>veg</sub> + RFP</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
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<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>
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<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>lepA</sub> + RFP</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">P<sub>lepA</sub> + RFP</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
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<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>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
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<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">High efficiency promoter and RBS for translation.</p></td>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<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>
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<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part produces a fungicide (antifungal peptide).</p></td>
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<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>
<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>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
<td><p class ="texte" style="padding-left: 10px; padding-top: 10px; padding-right: 10px; text-align:center;">We made it</p></td>
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<td><p class ="texte" style="padding-top: 10px; padding-left: 10px; padding-right: 10px;">This part is an integrative vector.</p></td>
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<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>
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<p class="title1" id="select4">Team safety</p>
<p class="title1" id="select4">Team safety</p>
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<p class="title2">Safety in INSA de Toulouse</p>
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<p class="title2">Safety in INSA Toulouse</p>
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<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>
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<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>
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>
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>
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>
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>
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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>
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>
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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>
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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>
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>
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>
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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>
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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>
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We were also informed about the potential chemical and microbiological risks thanks to her.<br>
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We were also informed about the potential chemical and microbiological risks.<br>
<br>
<br>
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The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedure of the LISBP.<br>
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The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedures of the LISBP.<br>
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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>
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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>
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).<br>
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).<br>
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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>
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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>
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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>
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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>
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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>
</p>
</p>
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<p class="citation">
<p class="citation">
"If you don't think it's safe, it probably isn't"</p>
"If you don't think it's safe, it probably isn't"</p>
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<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>
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<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>
<p class="title3">Personal protective equipment</p>
<p class="title3">Personal protective equipment</p>
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<p class="title3">Basic rules in a lab</p>
<p class="title3">Basic rules in a lab</p>
<p class="texte">We have to apply the basic safety principles into a laboratory room:<br>
<p class="texte">We have to apply the basic safety principles into a laboratory room:<br>
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- It is forbidden to smoke in all the rooms.<br>
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- It is forbidden to smoke in all rooms.<br>
- It is forbidden to drink and eat in the laboratory rooms.<br>
- It is forbidden to drink and eat in the laboratory rooms.<br>
- It is compulsory to wear a closed lab coat in cotton.<br>
- It is compulsory to wear a closed lab coat in cotton.<br>
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- Oral pipetting of any substance is prohibited in any laboratory.<br>
- Oral pipetting of any substance is prohibited in any laboratory.<br>
<br>
<br>
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There is also others precaution when working with biological organisms:<br>
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There are also others precautions when working with biological organisms:<br>
- We need to wash our hands regularly.<br>
- We need to wash our hands regularly.<br>
- It is compulsory to wear gloves except with the use of an electric burner.<br>
- It is compulsory to wear gloves except with the use of an electric burner.<br>
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.<br>
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.<br>
<br>
<br>
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Indeed, different machine are used to keep a sterile area.</p>
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Different apparatus are used to work a sterile area.</p>
<p class="title3">Waste</p>
<p class="title3">Waste</p>
<p class="texte">Different trash containers are available in the lab:<br>
<p class="texte">Different trash containers are available in the lab:<br>
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- One for biological waste (yellow). This waste will be autoclaved.<br>
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- One for biological waste (yellow). This waste will be autoclaved before being thrown out.<br>
<img width="150px" src="http://2014.igem.org/wiki/images/f/f7/Yellow_trash.JPG"><br><br>
<img width="150px" src="http://2014.igem.org/wiki/images/f/f7/Yellow_trash.JPG"><br><br>
- One for common waste (green or orange).<br>
- One for common waste (green or orange).<br>
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<p class="title4">Electric burner</p>
<p class="title4">Electric burner</p>
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<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>
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<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>
<p class="title4">Chemical hood</p>
<p class="title4">Chemical hood</p>
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<p class="title4">Water-bathes</p>
<p class="title4">Water-bathes</p>
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<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>
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<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>
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- risks to the safety and health of the general public,<br>
- risks to the safety and health of the general public,<br>
- risks to the environment,<br>
- risks to the environment,<br>
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- risks to security through malicious mis-use by individuals, groups, or countries.<br>
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- risks to security through malicious use by individuals, groups, or countries.<br>
<br>
<br>
There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).<br>
There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).<br>
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<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>
<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>
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.<br>
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.<br>
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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>
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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>
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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>
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>
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<p class="title3">How to reduce these Future risks?</p>
<p class="title3">How to reduce these Future risks?</p>
<p class="texte">We have thought about several ways to minimize risks: <br>
<p class="texte">We have thought about several ways to minimize risks: <br>
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- 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>
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- 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>
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- 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>
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- 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>
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- we want to use a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and another wild-type bacteria.</p>
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- we can build a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and any other wild-type bacteria.</p>
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Revision as of 20:17, 17 October 2014