Difference between revisions of "Team:Paris Bettencourt/Safety"

 
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Notebook Assay 15th August
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<td><a href="#ancre">Notebook</a></td>
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<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Protocols">Protocols</a></td>
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                                <td><a href="#ancre2">Assay</a></td>
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<td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Microbiology">Microbiology</a></td>
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<h1 class="red">Week 4th - 10th July</h1>
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<h3>Press wax on cellulose paper</h3>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/a/a0/Paris_Bettencourt-Notebook_Assay_Template.jpg" alt=« Template for wax » height=“150px“ />
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We used candle wax (vanilla scented) from Franprix. Wax was melted at 95 degrees. The 96 well plate template was used to create wax impressions on the cotton. Since wax is hydrophobic and the cotton we have in the lab is hydrophobic, it spreaded quickly, the diffusion was fast which makes the cotton unusable to carry out experiments or assay development.<br>
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<br>
 
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Cellulose paper for chromatography was used to carry out further experiments for assay developement. The template dipped in melted wax is pressed against the paper for sometime. We created successful impressions of wax on the paper where the wax penetrates vertically into the paper and creating specific boundaries. At times, we had to reheat the template pressed against the paper to further improve the penetration (but no longer than 10 secs at 100degrees). We could successfully recreate the assay on the paper. The wells are not even given the impression and dipping the template were done manually.<br>
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<!--h1 class="red">Lab safety </h1-->
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<br>
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Anthocyanin drops of volume 20ùl, 10ùl, 2ùl and 1ùl were used to test the wells. The Anthocyanin drops did not spread  and they were contained in the wells. We think 10ul can be used to perform experiments on cellulose paper. For experiments on assay, we need cotton which is neutral in nature.
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<div id="input">
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<h2 class="red">Summary</h2>
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<p>
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In iGEM we take safety seriously. Our project touches sensible topics such as enzyme and human contact so we had a special consideration about safety issues. From strict lab rules to interviews with drycleaners, we tried to measure user's awareness and potential problems in the implementation and safety of our final engineered GMO (genetically modified microorganism).
 
</p>
 
</p>
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<h2 class="red">Safety Training and Lab Rules</h2>
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<div id="figurebox">
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<img src="https://static.igem.org/mediawiki/2016/f/f0/Paris_Bettencourt-File_vue_cochin.jpg" alt="Quercetin strains degradation" style="width:900px;">
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<p>
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<b> Figure 1: Our laboratory facilities at the Cochin institut in Paris.</b>
 
</div>
 
</div>
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<br>
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<p>
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<br>
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At the beginning of the competition, all team members received a safety training for working in the Biosafety Level 1 and in the Biosafety level 2 laboratories. This training was conducted by the Biosafety officer for INSERM U1001 (Fig. 1) and it included best laboratory practices as described in the WHO laboratory biosafety manual.<br>
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<div class="input">
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It included rules about protocols in the lab, protective clothing and equipment, decontamination methods and practices, prevention of the transfer of genetic material and microorganisms, handling chemicals, use of machines and emergency procedures. The safety form was submitted to iGEM HQ as well.
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<h1 class="red">Week 18th -24th July</h1>
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<h3>Pressing wax on cotton fabric</h3>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/2/24/Paris_Bettencourt-Notebook_Assay_Wax_CNC.jpg" alt=« Wax CNC" />
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<p class=”input”>  
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We are still continuing research on wax, we have sent emails to the Musée Grévin, wax specialist, and iMakr that seemed to have printed candles in the shape of celebrities.
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<a href="https://3dprint.com/22463/scandles-bust-3d-print/">Scandles, 3D printed candles</a><br>
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In the meantime, we used the CNC (Computer Numeric Control) on a block of wax we've found in the Openlab we work in, to make 96 holes. The aim is to heat it and press it on cotton fabric to create circles in which we could poor liquid without it spreading to one another. We made the model in 3D for the CNC and it really worked well, wax is soft so it was easy but still quite long, approximately 3 hours. <br>
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</p>
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<h3>A mould to pour wax on frabric</h3>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/4/4c/Paris_Bettencourt-Notebook_Assay_Wax_mould.jpg" alt=«mould for wax» height=“150px“ />
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<img class="assay" src="https://static.igem.org/mediawiki/2016/c/c4/Paris_Bettencourt-Notebook_Assay_Wax_mould2.jpg" alt=« mould for wax » height=“150px“ />
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<img class="assay" src="https://static.igem.org/mediawiki/2016/c/cc/Paris_Bettencourt-Notebook_Assay_Wax_mould_piece.jpg" alt=« piece of mould for wax" height=“150px“ />
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<p class=”input”>
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We also decided to make a mould to poor wax in so that it would create circles of wax on cotton. We created a mould in solidworks to 3D print it. We first did it in one whole piece and the 3D print was not possible because some parts didn't have support so we then divided it into two parts that we 3D printed separately.  The pillars in the model were very tall, and consequently very fragile, two of them broke easily as one of our teammate played with it.
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<br>
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<div class="input">
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<h1 class="red">Week 25th -31th July</h1>
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<p class=”input”> This week we had a good talk with our advisor, Edwin, and realised together it would be much more interesting if we created an actual 96-well plate, that would be cheap and standardised to test products directly on fabric. So we are going to change our way of thinking from 2D to 3D. </p> <br>
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<h3>Using 3D printed wax on fabric</h3>
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<p class=”input”>
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First we still are looking options with wax as we saw wax can be 3D printed, indeed we've found a wax filament. To know more about it we've planned a skype call with Arthur Dalaise, one of our teammate's cousin that is founding a 3D printer service startup. The good news is that he agreed to try out printing with this wax filament for free and gave us really good advice ! <br>
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Our only concern with 3D printing is that we don't know if wax will stick to the fabric, not burn it and make real wells that don't allow liquid to spread.  
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</p>
 
</p>
  
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<h3>Using PDMS to create our own 96-well plate</h3>
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<h2 class="red">Biosafety Level 2</h2>
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<p class=”input”>  
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<p>
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Teja had a revelation and thought of using PDMS, a silicone-based polymer, to create a 96-well plate where we would sandwich a layer of PDMS with holes, cotton fabric, another layer of PDMS with holes and glass to be able to scan it.
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During this project we aimed, amongst other things, to create a database of strains isolated from vineyard soils from all around the world. Since we were handeling samples without knowing the microbes that were present on them, we took special security measurements. To begin with, we worked in a lab with biosafety level 2, in the appropriated hood and wearing the appropriate safety clothing. We also chose to work with a set of media that would be selective for non-pathogenical species, not allowing the potentially dangerous species that were on the samples to grow.
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</p>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/2/2b/Paris_Bettencourt-Notebook_Assay_PDMS_first_try.jpg" alt=« first try of PDMS assay" height=“150px“ />
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<p class=”input”>
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Our first try above is very promising, PDMS was punched by Teja to create the wells, so it is not perfect and doesn't fit well, we have to find ways to have a precise result. The cotton and PDMS don't stick together as well as we want them to.  
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</p>
 
</p>
  
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<h3>A 3D printed mould for PDMS</h3>
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<h2 class="red">Organisms used</h2>
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<p class=”input”> We decided to 3D print a mould with pillars to pour PDMS on it and have a layer of PDMS with holes in it. The pillars are 1cm high but half of it is slightly conic so that we can remove the PDMS easier.  
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<p>
 +
Our sole chassis organism in this project was <i>E. coli</i>. All cloning steps were performed in the DH5alpha strain, and all protein expression steps on the BL21(DE3) strain.<br>
 +
In the making of our database we isolated several different species of Acidovorax, Actinomycetes, Arthrobacter, Bacillus, Brevibacterium, Celullosimicrobium, Corynebacterium, Escherichia, Flavobacterium, Klebsiella, Kocuria, Microbacterium, Micrococcus, Oerskavia, Paenibacillus, Pantoea, Pseudomonas, Rhodococcus, Staphylococcus (non aureus) and Streptomyces. All our samples were sent for sequencing after growing them in selective media and no harmful species for human health was isolated from them.
 
</p>
 
</p>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/d/de/Paris_Bettencourt-Notebook_Assay_PDMS_first_mould.jpg " alt=« PDMS' first mould" height=“150px“ />
 
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<img class="assay" src="https://static.igem.org/mediawiki/2016/3/37/Paris_Bettencourt-Notebook_Assay_PDMS_first_fail_Teja.jpg" alt=« PDMS first failure" height=“150px“ />
 
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<img class="assay" src="https://static.igem.org/mediawiki/2016/7/71/Paris_Bettencourt-Notebook_Assay_PDMS_first_fail_part.jpg" alt=« part of PDMS fail" height=“150px“ />
 
  
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<p class=”input”>So this mould didn't work as well as planned ... The PDMS layer really hard to remove from the mould and we broke it and the mould while removing it. But the wells created on the parts that were not broken are really nice so we will do another try with a different mould with pillars that won't be as high.
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<h2 class="red">References</h2>
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</p>
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<ul>
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<br>
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<li>Public Health Statement for Tetrachloroethylene (PERC) from the Agency for Toxic Substances & Disease Registry of the United States Department of Health and Human Services (2014)
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<br>
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<h1 class="red">Week 1st - 7th August</h1>
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<h3>A new try for PDMS' 3D printed mould</h3>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/d/db/Paris_Bettencourt-Notebook_Assay_PDMS_mould_model.jpg" alt=« 3D model for PDMS mould" height=“150px“ />
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<p class=”input”>
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On this picture you can see our new 3D model with smaller pillars the total height is around 6mm. We are very confident with this model.
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</p>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/9/96/Paris_Bettencourt-Notebook_Assay_PDMS_layer.jpg" alt=« PDMS with holes" height=“150px“ />
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<p class=”input”>
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And this is the result of the 3D printing that went really well this time, we did a 80% infill so that the pillars don't break this time. The result of the PDMS is on the left, it is really what we expected and we are really happy with this. 
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</p>
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<h3>Lasercutting Cotton Fabric</h3>
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</li>
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<p class=”input”>
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We also decided to laser-cut cotton, our research on internet have shown it is already done and seems very efficient. We want to have circles of cotton that are linked so we don't have to put each circles of cotton into each wells. It worked really well and we managed to lasercut multiple layers at the same time (around 8).
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</p>
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<img class="assay" src="https://static.igem.org/mediawiki/2016/4/4b/Paris_Bettencourt-Notebook_Assay_Lasecut_Cotton.jpg" alt=« Lasercut Cotton" height=“150px“ />
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<p class=”input”>
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The borders of the lasercut cotton is then a bit burnt, which was totally expected but we fear it may interfere so we just soaked it in water and it disapeared instantly but the fabric shreds slightly after being wet.
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</p>
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<h3>Combining the different layers</h3>
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</div> <! End input Div>  
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<img class="assay" src="https://static.igem.org/mediawiki/2016/9/91/Paris_Bettencourt-Notebook_Assay_Layering_PDMS.jpg" alt=« Layering cotton and PDMS" height=“150px“ />
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</div> <! End subheader Div> 
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The cotton seems to fit really well, but we tried to stick the PDMS, cotton and glass layers on a small scale and it was not very good yet, the liquid spreads because the cotton is creating a small gap between the glass and the PDMS.
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</p>
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Thanks to Arthur Dalaise who helped us we have a functional assay using 3D printed wax filament on cotton. The first layers were printed with heated platform (100°C) with a 190°C temperature for the extruder and then the sheet of fabric was laid on, and finally the rest of the layers were printed on top of this with a unheated platform and a very hot extruder (240°C). The first step lasts for 30 mins and the second one for 6 hours. So the result is exactly what we want but the time it takes is really too long, so we will still continue the PDMS experiments.
 
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Latest revision as of 17:42, 19 October 2016




Summary

In iGEM we take safety seriously. Our project touches sensible topics such as enzyme and human contact so we had a special consideration about safety issues. From strict lab rules to interviews with drycleaners, we tried to measure user's awareness and potential problems in the implementation and safety of our final engineered GMO (genetically modified microorganism).

Safety Training and Lab Rules

Quercetin strains degradation

Figure 1: Our laboratory facilities at the Cochin institut in Paris.

At the beginning of the competition, all team members received a safety training for working in the Biosafety Level 1 and in the Biosafety level 2 laboratories. This training was conducted by the Biosafety officer for INSERM U1001 (Fig. 1) and it included best laboratory practices as described in the WHO laboratory biosafety manual.
It included rules about protocols in the lab, protective clothing and equipment, decontamination methods and practices, prevention of the transfer of genetic material and microorganisms, handling chemicals, use of machines and emergency procedures. The safety form was submitted to iGEM HQ as well.

Biosafety Level 2

During this project we aimed, amongst other things, to create a database of strains isolated from vineyard soils from all around the world. Since we were handeling samples without knowing the microbes that were present on them, we took special security measurements. To begin with, we worked in a lab with biosafety level 2, in the appropriated hood and wearing the appropriate safety clothing. We also chose to work with a set of media that would be selective for non-pathogenical species, not allowing the potentially dangerous species that were on the samples to grow.

Organisms used

Our sole chassis organism in this project was E. coli. All cloning steps were performed in the DH5alpha strain, and all protein expression steps on the BL21(DE3) strain.
In the making of our database we isolated several different species of Acidovorax, Actinomycetes, Arthrobacter, Bacillus, Brevibacterium, Celullosimicrobium, Corynebacterium, Escherichia, Flavobacterium, Klebsiella, Kocuria, Microbacterium, Micrococcus, Oerskavia, Paenibacillus, Pantoea, Pseudomonas, Rhodococcus, Staphylococcus (non aureus) and Streptomyces. All our samples were sent for sequencing after growing them in selective media and no harmful species for human health was isolated from them.

References

  • Public Health Statement for Tetrachloroethylene (PERC) from the Agency for Toxic Substances & Disease Registry of the United States Department of Health and Human Services (2014)
Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com
2016.igem.org