http://2014.igem.org/wiki/index.php?title=Special:Contributions/Seafloor&feed=atom&limit=50&target=Seafloor&year=&month=2014.igem.org - User contributions [en]2024-03-28T16:58:28ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:ReadingTeam:Reading2014-10-18T01:10:52Z<p>Seafloor: </p>
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<p>Biological photovoltaics (<a href="https://en.wikipedia.org/wiki/Biological_photovoltaics">BPVs</a>) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, <i>Synechocystis</i> sp. PCC 6803. </p><br />
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<td width="40%" align="right"><br />
<p>Read more on our <a href="https://2014.igem.org/Team:Reading/Project">Project</a> page.</p><br />
<p>If you'd like to contact the team, don't hesitate to get in touch!</br><br />
Twitter: <a href="https://twitter.com/RUiGEMTeam">@RUiGEMTeam</a></br><br />
Email: <a href="mailto:rusynbioigem@gmail.com">rusynbioigem@gmail.com</a></br><br />
Facebook: <a href="https://www.facebook.com/RUIGEM2014">RUiGEM</a></p></br> <br />
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<h3><b>School of Systems Engineering,<br />
<br>University of Reading</b></h3><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:ReadingTeam:Reading2014-10-18T01:10:29Z<p>Seafloor: </p>
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<p>Biological photovoltaics (<a href="https://en.wikipedia.org/wiki/Biological_photovoltaics">BPVs</a>) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, <i>Synechocystis</i> sp. PCC 6803. </p><br />
</br></br><br />
</td><br />
<td width="40%" align="right"><br />
<p>Read more on our <a href="https://2014.igem.org/Team:Reading/Project">Project</a> page.</p><br />
<p>If you'd like to contact the team, don't hesitate to get in touch!</br><br />
Twitter: <a href="https://twitter.com/RUiGEMTeam">@RUiGEMTeam</a></br><br />
Email: <a href="mailto:rusynbioigem@gmail.com">rusynbioigem@gmail.com</a></br><br />
Facebook: <a href="https://www.facebook.com/RUIGEM2014">RUiGEM</a></p></br> <br />
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<h3><b>School of Systems Engineering,<br />
<br>University of Reading</b></h3><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:ReadingTeam:Reading2014-10-18T01:10:09Z<p>Seafloor: </p>
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<h3 class="title">Project Overview</h3><br />
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<table><br />
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<td align="left"><br />
<p>Biological photovoltaics (<a href="https://en.wikipedia.org/wiki/Biological_photovoltaics">BPVs</a>) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, <i>Synechocystis</i> sp. PCC 6803. </p><br />
</br></br><br />
</td><br />
<td width="40%" align="right"><br />
<p>Read more on our <a href="https://2014.igem.org/Team:Reading/Project">Project</a> page.</p><br />
<p>If you'd like to contact the team, don't hesitate to get in touch!</br><br />
Twitter: <a href="https://twitter.com/RUiGEMTeam">@RUiGEMTeam</a></br><br />
Email: <a href="mailto:rusynbioigem@gmail.com">rusynbioigem@gmail.com</a></br><br />
Facebook: <a href="https://www.facebook.com/RUIGEM2014">RUiGEM</a></p></br> <br />
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<h3><b>School of Systems Engineering,<br />
<br>University of Reading</b></h3><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:ReadingTeam:Reading2014-10-18T01:09:42Z<p>Seafloor: </p>
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<p>Biological photovoltaics (<a href="https://en.wikipedia.org/wiki/Biological_photovoltaics">BPVs</a>) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, <i>Synechocystis</i> sp. PCC 6803. </p><br />
</br></br><br />
</td><br />
<td width="40%" align="right"><br />
<p>Read more on our <a href="https://2014.igem.org/Team:Reading/Project">Project</a> page.</p><br />
<p>If you'd like to contact the team, don't hesitate to get in touch!</br><br />
Twitter: <a href="https://twitter.com/RUiGEMTeam">@RUiGEMTeam</a></br><br />
Email: <a href="mailto:rusynbioigem@gmail.com">rusynbioigem@gmail.com</a></br><br />
Facebook: <a href="https://www.facebook.com/RUIGEM2014">RUiGEM</a></p></br> <br />
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<a href="http://www.snapgene.com/"><img src="https://static.igem.org/mediawiki/2014/9/9e/Tue2014_Sponsors_SnapGene.png" width="60%" hspace="40" /></a><br />
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<h3><b>School of Systems Engineering,<br />
<br>University of Reading</b></h3><br />
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<a href="http://http://www.citeab.com/"><img src="https://static.igem.org/mediawiki/2014/d/dc/Cite_AB_Logo.png" width="40%" hspace="40" /></a><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:ReadingTeam:Reading2014-10-18T01:09:20Z<p>Seafloor: </p>
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<h3 class="title">Project Overview</h3><br />
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<table><br />
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<td align="left"><br />
<p>Biological photovoltaics (<a href="https://en.wikipedia.org/wiki/Biological_photovoltaics">BPVs</a>) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, <i>Synechocystis</i> sp. PCC 6803. </p><br />
</br></br><br />
</td><br />
<td width="40%" align="right"><br />
<p>Read more on our <a href="https://2014.igem.org/Team:Reading/Project">Project</a> page.</p><br />
<p>If you'd like to contact the team, don't hesitate to get in touch!</br><br />
Twitter: <a href="https://twitter.com/RUiGEMTeam">@RUiGEMTeam</a></br><br />
Email: <a href="mailto:rusynbioigem@gmail.com">rusynbioigem@gmail.com</a></br><br />
Facebook: <a href="https://www.facebook.com/RUIGEM2014">RUiGEM</a></p></br> <br />
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<a href="http://www.snapgene.com/"><img src="https://static.igem.org/mediawiki/2014/9/9e/Tue2014_Sponsors_SnapGene.png" width="60%" hspace="40" /></a><br />
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<h3><b>School of Systems Engineering,<br />
<br>University of Reading</b></h3><br />
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<a href="http://http://www.citeab.com/"><img src="https://static.igem.org/mediawiki/2014/d/dc/Cite_AB_Logo.png" width="40%" hspace="40" /></a><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/ProtocolsTeam:Reading/Protocols2014-10-18T00:58:49Z<p>Seafloor: </p>
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<td><h3 class="title">A note on protocols</h3></td><br />
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<td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p><br />
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<h3 class="title">Contents</h3><br />
<ol><br />
<li><a href="#">A Note on Protocols</a><br />
<li><a href="#protocols">Protocols</a><br />
<ul><br />
<li><a href="#prot1">Miniprep</a><br />
<li><a href="#prot2">Glycerol stock</a><br />
<li><a href="#prot3">Optical Density</a><br />
<li><a href="#prot4">PCR</a><br />
<li id=protocols><a href="#prot5">Transformation (E. coli)</a><br />
<li><a href="#prot6">Nanodrop</a><br />
<li id=prot1><a href="#prot7">BG-11 plates</a><br />
<li><a href="#prot8">Cyanobacteria Transformation</a><br />
<li><a href="#prot9">Biobrick Assembly</a></ul><br />
</ul><br />
<li><a href="#references">References</a><br />
<li><a href="#acknowledge">Acknowledgements</a><br />
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<h3 class="title">Protocols</h3><br />
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<!--Miniprep --><br />
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<p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p><br />
<ol><br />
<li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes<br />
<li>Discard supernatant by pipetting off ensuring not to disturb the pellet<br />
<li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes<br />
<li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times<br />
<li>Centrifuge at 13,000 rpm for 5 minutes<br />
<li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate<br />
<li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm<br />
<li>Discard the flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li id=prot2>Discard flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Discard flow through<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube<br />
<li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette<br />
<li>Incubate at room temperature for 2 minutes<br />
<li>Centrifuge for 2 minutes at 13,000 rpm<br />
</ol><br />
<br />
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<br><br />
<p class="title"><i>Making glycerol stock</i></p><br />
<p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p><br />
<ol><br />
<li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips<br />
<li>Centrifuge at 13,000 rpm for 2 minutes<br />
<li>Discard supernatant<br />
<li>Add 0.5 ml of 60% glycerol stock<br />
<ol style="list-style: lower-roman outside"><br />
<li>240 ml of glycerol<br />
<li>160 ml nano pure water<br />
<li>mix together and autoclave<br />
</ol><br />
<li>freeze at -80℃<br />
</ol><br />
<br />
<!-- Optical Density --><br />
<br><br />
<p class="title"><i>Taking optical density of culture</i></p><br />
<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p><br />
<ol><br />
<li>Set spectrophotometer to measure at OD<sub>750</sub><br />
<li>Blank with 1 ml of BG-11 in a cuvette<br />
<li>Measure 1 ml of culture<br />
<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11<br />
</ol><br />
<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p><br />
<br />
<!-- PCR --><br />
<br><br />
<p class="title"><i>PCR</i></p><br />
<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p><br />
<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p><br />
<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p><br />
<p>PCR program program as follows:</p><br />
<ol><br />
<li> Start:<br />
<ul><br />
<li>95℃ for 30 seconds<br />
</ul><br />
<li id=prot5>35 cycles:<br />
<ul><br />
<li>95℃ for 10 seconds<br />
<li>56℃ for 15 seconds<br />
<li>72℃ for 70 seconds<br />
</ul><br />
<li>Final<br />
<ul><br />
<li>72℃ for 5 minutes<br />
<li>68℃ for 10 minutes<br />
</ul><br />
</ol><br />
<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p><br />
<br />
<!-- Transformation (E. coli) --><br />
<br><br />
<p class="title"><i>Transformation into E. coli</i></p><br />
<p><br />
<ol><br />
<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf<br />
<li>Add 5 ul of DNA using a sterile pipette tip<br />
<li>Flick the tubes to mix and then store on ice for 30 minutes<br />
<li>Heat shock in a water bath at 42℃ for 1 minute<br />
<li>Incubate on ice for 5 minutes<br />
<li id=prot6>Add 450 ul of SOC (we used LB instead)<br />
<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm<br />
<li>Invert Eppendorfs containing the cells several times<br />
<li>Plate out and incubate overnight at 37℃<br />
</ol><br />
<p> Notes on transformation efficency</p><br />
<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). <a id=prot7 />We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p><br />
<br />
<!-- Nanodrop --><br />
<br><br />
<p class="title"><i>Nanodrop</i></p><br />
<p>Nanodrop is used to check concentration in DNA often from a miniprep</p><br />
<ol><br />
<li>Set the Nanodrop to measure DNA<br />
<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank<br />
<li>Add 2 ul of sample and measure. Measurement should be in ng/ul<br />
</ol><br />
<br />
<!-- BG-11 plates --><br />
<br><br />
<p class="title"><i>BG-11 plates</i></p><br />
<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p><br />
<ol><br />
<li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise<br />
<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml<br />
<li>Agar melted in steamer and kept at 55℃ until needed for pouring<br />
</ol><br />
<p> If making a kanamycin cap:</p><br />
<ol><br />
<li>0.6% agar w/v<br />
<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan<br />
<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it<br />
<li>Leave for >1 week for Kan selection to occur<br />
</ol><br />
<br />
<!-- Cyanobacteria Transformation --><br />
<br><br />
<p class="title"><i>Cyanobacteria Transformation</i></p><br />
<p>Protocol to transform DNA into cyanobacteria</p><br />
<ol><br />
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days<br />
<li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant<br />
<li>Repeat step 2<br />
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant<br />
<li>Add 200ml fresh BG-11<br />
<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)<br />
<li>Incubate for 24 under light on a shaker at ~100 rpm<br />
<li>Plate the full 200ml and leave for 1-2 days<br />
<li>Add 3-4ml kan 0.6% agar BG-11<br />
<li>Leave under light for >1 week<br />
</ol><br />
<p> After many attempts to transform with Cyanobacteria we refined this protocol<br />
<ol><br />
<li>Grow 6803 to ~0.435<br />
<li>Take 1 ml, re-wash with BG-11<br />
<li>Re-suspend in 90ml BG-11<br />
<li>Plasmid DNA at ~270ng/ul<br />
<li>add 8ul to each tube<br />
<li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes<br />
<li>transfer to warm room at 28ºC and leave overnight<br />
<li>plate on Kan50 and leave under light, agar-side down<br />
</ol><br />
<br />
<!-- Biobrick Assembly --><br />
<br><br />
<p class="title"><i>Biobrick Assembly</i></p><br />
<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p><br />
<ol><br />
<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone<br />
<li>To the same tube add 2.5 ul of NED buffer<br />
<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1<br />
<li>Incubate the tubes at 37℃ for 15 minutes<br />
<li>Then incubate at 80℃ for 20 minutes <br />
<li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone<br />
<li>Add 2 ul of 10X T4 DNA ligase restriction buffer<br />
<li>Add 1 ul of T4 DNA ligase <br />
<li>Incubate at room temperature for 10 minutes <br />
<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme<br />
</ol><br />
<p>This DNA can now be used for transformation</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title">References</h3><br />
<p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p><br />
<p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p><br />
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p><br />
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p><br />
<p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p><br />
<p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p><br />
</td><br />
</tr><br />
</table><br />
</html><br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/File:Oscar_doing_stuff.jpgFile:Oscar doing stuff.jpg2014-10-18T00:58:32Z<p>Seafloor: uploaded a new version of &quot;File:Oscar doing stuff.jpg&quot;</p>
<hr />
<div></div>Seafloorhttp://2014.igem.org/Team:Reading/ProtocolsTeam:Reading/Protocols2014-10-18T00:54:19Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br />
<table><br />
<br />
<!--Protocols --><br />
<tr><br />
<td><h3 class="title">A note on protocols</h3></td><br />
</tr><br />
<br />
<!-- Introduction to the Protocols Page --><br />
<tr><br />
<td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p><br />
<br><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/0/0d/Oscar_doing_stuff.jpg" width="600"/></p><br />
</td><br />
<br />
<!-- Contents --><br />
<td width="27%" valign="top"><br />
<h3 class="title">Contents</h3><br />
<ol><br />
<li><a href="#">A Note on Protocols</a><br />
<li><a href="#protocols">Protocols</a><br />
<ul><br />
<li><a href="#prot1">Miniprep</a><br />
<li><a href="#prot2">Glycerol stock</a><br />
<li><a href="#prot3">Optical Density</a><br />
<li><a href="#prot4">PCR</a><br />
<li id=protocols><a href="#prot5">Transformation (E. coli)</a><br />
<li><a href="#prot6">Nanodrop</a><br />
<li id=prot1><a href="#prot7">BG-11 plates</a><br />
<li><a href="#prot8">Cyanobacteria Transformation</a><br />
<li><a href="#prot9">Biobrick Assembly</a></ul><br />
</ul><br />
<li><a href="#references">References</a><br />
<li><a href="#acknowledge">Acknowledgements</a><br />
</ol><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr><td colspan="3" height="15px"></td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"></tr><br />
<tr><td colspan="3" height="5px"></td></tr><br />
<br />
<!-- The Protocols --><br />
<tr><br />
<td colspan="3"><br />
<h3 class="title">Protocols</h3><br />
<br />
<!--Miniprep --><br />
<br><br />
<p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p><br />
<ol><br />
<li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes<br />
<li>Discard supernatant by pipetting off ensuring not to disturb the pellet<br />
<li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes<br />
<li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times<br />
<li>Centrifuge at 13,000 rpm for 5 minutes<br />
<li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate<br />
<li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm<br />
<li>Discard the flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li id=prot2>Discard flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Discard flow through<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube<br />
<li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette<br />
<li>Incubate at room temperature for 2 minutes<br />
<li>Centrifuge for 2 minutes at 13,000 rpm<br />
</ol><br />
<br />
<!-- Glycerol stock --><br />
<br><br />
<p class="title"><i>Making glycerol stock</i></p><br />
<p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p><br />
<ol><br />
<li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips<br />
<li>Centrifuge at 13,000 rpm for 2 minutes<br />
<li>Discard supernatant<br />
<li>Add 0.5 ml of 60% glycerol stock<br />
<ol style="list-style: lower-roman outside"><br />
<li>240 ml of glycerol<br />
<li>160 ml nano pure water<br />
<li>mix together and autoclave<br />
</ol><br />
<li>freeze at -80℃<br />
</ol><br />
<br />
<!-- Optical Density --><br />
<br><br />
<p class="title"><i>Taking optical density of culture</i></p><br />
<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p><br />
<ol><br />
<li>Set spectrophotometer to measure at OD<sub>750</sub><br />
<li>Blank with 1 ml of BG-11 in a cuvette<br />
<li>Measure 1 ml of culture<br />
<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11<br />
</ol><br />
<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p><br />
<br />
<!-- PCR --><br />
<br><br />
<p class="title"><i>PCR</i></p><br />
<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p><br />
<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p><br />
<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p><br />
<p>PCR program program as follows:</p><br />
<ol><br />
<li> Start:<br />
<ul><br />
<li>95℃ for 30 seconds<br />
</ul><br />
<li id=prot5>35 cycles:<br />
<ul><br />
<li>95℃ for 10 seconds<br />
<li>56℃ for 15 seconds<br />
<li>72℃ for 70 seconds<br />
</ul><br />
<li>Final<br />
<ul><br />
<li>72℃ for 5 minutes<br />
<li>68℃ for 10 minutes<br />
</ul><br />
</ol><br />
<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p><br />
<br />
<!-- Transformation (E. coli) --><br />
<br><br />
<p class="title"><i>Transformation into E. coli</i></p><br />
<p><br />
<ol><br />
<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf<br />
<li>Add 5 ul of DNA using a sterile pipette tip<br />
<li>Flick the tubes to mix and then store on ice for 30 minutes<br />
<li>Heat shock in a water bath at 42℃ for 1 minute<br />
<li>Incubate on ice for 5 minutes<br />
<li id=prot6>Add 450 ul of SOC (we used LB instead)<br />
<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm<br />
<li>Invert Eppendorfs containing the cells several times<br />
<li>Plate out and incubate overnight at 37℃<br />
</ol><br />
<p> Notes on transformation efficency</p><br />
<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). <a id=prot7 />We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p><br />
<br />
<!-- Nanodrop --><br />
<br><br />
<p class="title"><i>Nanodrop</i></p><br />
<p>Nanodrop is used to check concentration in DNA often from a miniprep</p><br />
<ol><br />
<li>Set the Nanodrop to measure DNA<br />
<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank<br />
<li>Add 2 ul of sample and measure. Measurement should be in ng/ul<br />
</ol><br />
<br />
<!-- BG-11 plates --><br />
<br><br />
<p class="title"><i>BG-11 plates</i></p><br />
<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p><br />
<ol><br />
<li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise<br />
<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml<br />
<li>Agar melted in steamer and kept at 55℃ until needed for pouring<br />
</ol><br />
<p> If making a kanamycin cap:</p><br />
<ol><br />
<li>0.6% agar w/v<br />
<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan<br />
<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it<br />
<li>Leave for >1 week for Kan selection to occur<br />
</ol><br />
<br />
<!-- Cyanobacteria Transformation --><br />
<br><br />
<p class="title"><i>Cyanobacteria Transformation</i></p><br />
<p>Protocol to transform DNA into cyanobacteria</p><br />
<ol><br />
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days<br />
<li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant<br />
<li>Repeat step 2<br />
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant<br />
<li>Add 200ml fresh BG-11<br />
<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)<br />
<li>Incubate for 24 under light on a shaker at ~100 rpm<br />
<li>Plate the full 200ml and leave for 1-2 days<br />
<li>Add 3-4ml kan 0.6% agar BG-11<br />
<li>Leave under light for >1 week<br />
</ol><br />
<p> After many attempts to transform with Cyanobacteria we refined this protocol<br />
<ol><br />
<li>Grow 6803 to ~0.435<br />
<li>Take 1 ml, re-wash with BG-11<br />
<li>Re-suspend in 90ml BG-11<br />
<li>Plasmid DNA at ~270ng/ul<br />
<li>add 8ul to each tube<br />
<li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes<br />
<li>transfer to warm room at 28ºC and leave overnight<br />
<li>plate on Kan50 and leave under light, agar-side down<br />
</ol><br />
<br />
<!-- Biobrick Assembly --><br />
<br><br />
<p class="title"><i>Biobrick Assembly</i></p><br />
<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p><br />
<ol><br />
<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone<br />
<li>To the same tube add 2.5 ul of NED buffer<br />
<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1<br />
<li>Incubate the tubes at 37℃ for 15 minutes<br />
<li>Then incubate at 80℃ for 20 minutes <br />
<li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone<br />
<li>Add 2 ul of 10X T4 DNA ligase restriction buffer<br />
<li>Add 1 ul of T4 DNA ligase <br />
<li>Incubate at room temperature for 10 minutes <br />
<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme<br />
</ol><br />
<p>This DNA can now be used for transformation</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title">References</h3><br />
<p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p><br />
<p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p><br />
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p><br />
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p><br />
<p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p><br />
<p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p><br />
</td><br />
</tr><br />
</table><br />
</html><br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/ProtocolsTeam:Reading/Protocols2014-10-18T00:54:04Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br />
<table><br />
<br />
<!--Protocols --><br />
<tr><br />
<td><h3 class="title">A note on protocols</h3></td><br />
</tr><br />
<br />
<!-- Introduction to the Protocols Page --><br />
<tr><br />
<td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p><br />
<br><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/0/0d/Oscar_doing_stuff.jpg" width="600"/></p><br />
</td><br />
<br />
<!-- Contents --><br />
<td width="27%" valign="top"><br />
<h3 class="title"> Contents</h3><br />
<ol><br />
<li><a href="#">A Note on Protocols</a><br />
<li><a href="#protocols">Protocols</a><br />
<ul><br />
<li><a href="#prot1">Miniprep</a><br />
<li><a href="#prot2">Glycerol stock</a><br />
<li><a href="#prot3">Optical Density</a><br />
<li><a href="#prot4">PCR</a><br />
<li id=protocols><a href="#prot5">Transformation (E. coli)</a><br />
<li><a href="#prot6">Nanodrop</a><br />
<li id=prot1><a href="#prot7">BG-11 plates</a><br />
<li><a href="#prot8">Cyanobacteria Transformation</a><br />
<li><a href="#prot9">Biobrick Assembly</a></ul><br />
</ul><br />
<li><a href="#references">References</a><br />
<li><a href="#acknowledge">Acknowledgements</a><br />
</ol><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr><td colspan="3" height="15px"></td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"></tr><br />
<tr><td colspan="3" height="5px"></td></tr><br />
<br />
<!-- The Protocols --><br />
<tr><br />
<td colspan="3"><br />
<h3 class="title">Protocols</h3><br />
<br />
<!--Miniprep --><br />
<br><br />
<p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p><br />
<ol><br />
<li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes<br />
<li>Discard supernatant by pipetting off ensuring not to disturb the pellet<br />
<li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes<br />
<li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times<br />
<li>Centrifuge at 13,000 rpm for 5 minutes<br />
<li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate<br />
<li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm<br />
<li>Discard the flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li id=prot2>Discard flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Discard flow through<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube<br />
<li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette<br />
<li>Incubate at room temperature for 2 minutes<br />
<li>Centrifuge for 2 minutes at 13,000 rpm<br />
</ol><br />
<br />
<!-- Glycerol stock --><br />
<br><br />
<p class="title"><i>Making glycerol stock</i></p><br />
<p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p><br />
<ol><br />
<li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips<br />
<li>Centrifuge at 13,000 rpm for 2 minutes<br />
<li>Discard supernatant<br />
<li>Add 0.5 ml of 60% glycerol stock<br />
<ol style="list-style: lower-roman outside"><br />
<li>240 ml of glycerol<br />
<li>160 ml nano pure water<br />
<li>mix together and autoclave<br />
</ol><br />
<li>freeze at -80℃<br />
</ol><br />
<br />
<!-- Optical Density --><br />
<br><br />
<p class="title"><i>Taking optical density of culture</i></p><br />
<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p><br />
<ol><br />
<li>Set spectrophotometer to measure at OD<sub>750</sub><br />
<li>Blank with 1 ml of BG-11 in a cuvette<br />
<li>Measure 1 ml of culture<br />
<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11<br />
</ol><br />
<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p><br />
<br />
<!-- PCR --><br />
<br><br />
<p class="title"><i>PCR</i></p><br />
<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p><br />
<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p><br />
<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p><br />
<p>PCR program program as follows:</p><br />
<ol><br />
<li> Start:<br />
<ul><br />
<li>95℃ for 30 seconds<br />
</ul><br />
<li id=prot5>35 cycles:<br />
<ul><br />
<li>95℃ for 10 seconds<br />
<li>56℃ for 15 seconds<br />
<li>72℃ for 70 seconds<br />
</ul><br />
<li>Final<br />
<ul><br />
<li>72℃ for 5 minutes<br />
<li>68℃ for 10 minutes<br />
</ul><br />
</ol><br />
<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p><br />
<br />
<!-- Transformation (E. coli) --><br />
<br><br />
<p class="title"><i>Transformation into E. coli</i></p><br />
<p><br />
<ol><br />
<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf<br />
<li>Add 5 ul of DNA using a sterile pipette tip<br />
<li>Flick the tubes to mix and then store on ice for 30 minutes<br />
<li>Heat shock in a water bath at 42℃ for 1 minute<br />
<li>Incubate on ice for 5 minutes<br />
<li id=prot6>Add 450 ul of SOC (we used LB instead)<br />
<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm<br />
<li>Invert Eppendorfs containing the cells several times<br />
<li>Plate out and incubate overnight at 37℃<br />
</ol><br />
<p> Notes on transformation efficency</p><br />
<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). <a id=prot7 />We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p><br />
<br />
<!-- Nanodrop --><br />
<br><br />
<p class="title"><i>Nanodrop</i></p><br />
<p>Nanodrop is used to check concentration in DNA often from a miniprep</p><br />
<ol><br />
<li>Set the Nanodrop to measure DNA<br />
<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank<br />
<li>Add 2 ul of sample and measure. Measurement should be in ng/ul<br />
</ol><br />
<br />
<!-- BG-11 plates --><br />
<br><br />
<p class="title"><i>BG-11 plates</i></p><br />
<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p><br />
<ol><br />
<li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise<br />
<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml<br />
<li>Agar melted in steamer and kept at 55℃ until needed for pouring<br />
</ol><br />
<p> If making a kanamycin cap:</p><br />
<ol><br />
<li>0.6% agar w/v<br />
<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan<br />
<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it<br />
<li>Leave for >1 week for Kan selection to occur<br />
</ol><br />
<br />
<!-- Cyanobacteria Transformation --><br />
<br><br />
<p class="title"><i>Cyanobacteria Transformation</i></p><br />
<p>Protocol to transform DNA into cyanobacteria</p><br />
<ol><br />
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days<br />
<li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant<br />
<li>Repeat step 2<br />
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant<br />
<li>Add 200ml fresh BG-11<br />
<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)<br />
<li>Incubate for 24 under light on a shaker at ~100 rpm<br />
<li>Plate the full 200ml and leave for 1-2 days<br />
<li>Add 3-4ml kan 0.6% agar BG-11<br />
<li>Leave under light for >1 week<br />
</ol><br />
<p> After many attempts to transform with Cyanobacteria we refined this protocol<br />
<ol><br />
<li>Grow 6803 to ~0.435<br />
<li>Take 1 ml, re-wash with BG-11<br />
<li>Re-suspend in 90ml BG-11<br />
<li>Plasmid DNA at ~270ng/ul<br />
<li>add 8ul to each tube<br />
<li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes<br />
<li>transfer to warm room at 28ºC and leave overnight<br />
<li>plate on Kan50 and leave under light, agar-side down<br />
</ol><br />
<br />
<!-- Biobrick Assembly --><br />
<br><br />
<p class="title"><i>Biobrick Assembly</i></p><br />
<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p><br />
<ol><br />
<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone<br />
<li>To the same tube add 2.5 ul of NED buffer<br />
<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1<br />
<li>Incubate the tubes at 37℃ for 15 minutes<br />
<li>Then incubate at 80℃ for 20 minutes <br />
<li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone<br />
<li>Add 2 ul of 10X T4 DNA ligase restriction buffer<br />
<li>Add 1 ul of T4 DNA ligase <br />
<li>Incubate at room temperature for 10 minutes <br />
<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme<br />
</ol><br />
<p>This DNA can now be used for transformation</p><br />
</td><br />
</tr><br />
<br />
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<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title">References</h3><br />
<p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p><br />
<p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p><br />
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p><br />
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p><br />
<p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p><br />
<p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p><br />
</td><br />
</tr><br />
</table><br />
</html><br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/ProtocolsTeam:Reading/Protocols2014-10-18T00:53:43Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br />
<table><br />
<br />
<!--Protocols --><br />
<tr><br />
<td><h3 class="title">A note on protocols</h3></td><br />
</tr><br />
<br />
<!-- Introduction to the Protocols Page --><br />
<tr><br />
<td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p><br />
<br><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/0/0d/Oscar_doing_stuff.jpg" width="600"/></p><br />
</td><br />
<br />
<!-- Contents --><br />
<td width="27%" valign="top"><br />
<h3 class="title">Contents</h3><br />
<ol><br />
<li><a href="#">A Note on Protocols</a><br />
<li><a href="#protocols">Protocols</a><br />
<ul><br />
<li><a href="#prot1">Miniprep</a><br />
<li><a href="#prot2">Glycerol stock</a><br />
<li><a href="#prot3">Optical Density</a><br />
<li><a href="#prot4">PCR</a><br />
<li id=protocols><a href="#prot5">Transformation (E. coli)</a><br />
<li><a href="#prot6">Nanodrop</a><br />
<li id=prot1><a href="#prot7">BG-11 plates</a><br />
<li><a href="#prot8">Cyanobacteria Transformation</a><br />
<li><a href="#prot9">Biobrick Assembly</a></ul><br />
</ul><br />
<li><a href="#references">References</a><br />
<li><a href="#acknowledge">Acknowledgements</a><br />
</ol><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr><td colspan="3" height="15px"></td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"></tr><br />
<tr><td colspan="3" height="5px"></td></tr><br />
<br />
<!-- The Protocols --><br />
<tr><br />
<td colspan="3"><br />
<h3 class="title">Protocols</h3><br />
<br />
<!--Miniprep --><br />
<br><br />
<p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p><br />
<ol><br />
<li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes<br />
<li>Discard supernatant by pipetting off ensuring not to disturb the pellet<br />
<li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes<br />
<li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times<br />
<li>Centrifuge at 13,000 rpm for 5 minutes<br />
<li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate<br />
<li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm<br />
<li>Discard the flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li id=prot2>Discard flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Discard flow through<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube<br />
<li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette<br />
<li>Incubate at room temperature for 2 minutes<br />
<li>Centrifuge for 2 minutes at 13,000 rpm<br />
</ol><br />
<br />
<!-- Glycerol stock --><br />
<br><br />
<p class="title"><i>Making glycerol stock</i></p><br />
<p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p><br />
<ol><br />
<li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips<br />
<li>Centrifuge at 13,000 rpm for 2 minutes<br />
<li>Discard supernatant<br />
<li>Add 0.5 ml of 60% glycerol stock<br />
<ol style="list-style: lower-roman outside"><br />
<li>240 ml of glycerol<br />
<li>160 ml nano pure water<br />
<li>mix together and autoclave<br />
</ol><br />
<li>freeze at -80℃<br />
</ol><br />
<br />
<!-- Optical Density --><br />
<br><br />
<p class="title"><i>Taking optical density of culture</i></p><br />
<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p><br />
<ol><br />
<li>Set spectrophotometer to measure at OD<sub>750</sub><br />
<li>Blank with 1 ml of BG-11 in a cuvette<br />
<li>Measure 1 ml of culture<br />
<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11<br />
</ol><br />
<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p><br />
<br />
<!-- PCR --><br />
<br><br />
<p class="title"><i>PCR</i></p><br />
<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p><br />
<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p><br />
<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p><br />
<p>PCR program program as follows:</p><br />
<ol><br />
<li> Start:<br />
<ul><br />
<li>95℃ for 30 seconds<br />
</ul><br />
<li id=prot5>35 cycles:<br />
<ul><br />
<li>95℃ for 10 seconds<br />
<li>56℃ for 15 seconds<br />
<li>72℃ for 70 seconds<br />
</ul><br />
<li>Final<br />
<ul><br />
<li>72℃ for 5 minutes<br />
<li>68℃ for 10 minutes<br />
</ul><br />
</ol><br />
<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p><br />
<br />
<!-- Transformation (E. coli) --><br />
<br><br />
<p class="title"><i>Transformation into E. coli</i></p><br />
<p><br />
<ol><br />
<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf<br />
<li>Add 5 ul of DNA using a sterile pipette tip<br />
<li>Flick the tubes to mix and then store on ice for 30 minutes<br />
<li>Heat shock in a water bath at 42℃ for 1 minute<br />
<li>Incubate on ice for 5 minutes<br />
<li id=prot6>Add 450 ul of SOC (we used LB instead)<br />
<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm<br />
<li>Invert Eppendorfs containing the cells several times<br />
<li>Plate out and incubate overnight at 37℃<br />
</ol><br />
<p> Notes on transformation efficency</p><br />
<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). <a id=prot7 />We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p><br />
<br />
<!-- Nanodrop --><br />
<br><br />
<p class="title"><i>Nanodrop</i></p><br />
<p>Nanodrop is used to check concentration in DNA often from a miniprep</p><br />
<ol><br />
<li>Set the Nanodrop to measure DNA<br />
<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank<br />
<li>Add 2 ul of sample and measure. Measurement should be in ng/ul<br />
</ol><br />
<br />
<!-- BG-11 plates --><br />
<br><br />
<p class="title"><i>BG-11 plates</i></p><br />
<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p><br />
<ol><br />
<li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise<br />
<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml<br />
<li>Agar melted in steamer and kept at 55℃ until needed for pouring<br />
</ol><br />
<p> If making a kanamycin cap:</p><br />
<ol><br />
<li>0.6% agar w/v<br />
<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan<br />
<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it<br />
<li>Leave for >1 week for Kan selection to occur<br />
</ol><br />
<br />
<!-- Cyanobacteria Transformation --><br />
<br><br />
<p class="title"><i>Cyanobacteria Transformation</i></p><br />
<p>Protocol to transform DNA into cyanobacteria</p><br />
<ol><br />
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days<br />
<li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant<br />
<li>Repeat step 2<br />
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant<br />
<li>Add 200ml fresh BG-11<br />
<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)<br />
<li>Incubate for 24 under light on a shaker at ~100 rpm<br />
<li>Plate the full 200ml and leave for 1-2 days<br />
<li>Add 3-4ml kan 0.6% agar BG-11<br />
<li>Leave under light for >1 week<br />
</ol><br />
<p> After many attempts to transform with Cyanobacteria we refined this protocol<br />
<ol><br />
<li>Grow 6803 to ~0.435<br />
<li>Take 1 ml, re-wash with BG-11<br />
<li>Re-suspend in 90ml BG-11<br />
<li>Plasmid DNA at ~270ng/ul<br />
<li>add 8ul to each tube<br />
<li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes<br />
<li>transfer to warm room at 28ºC and leave overnight<br />
<li>plate on Kan50 and leave under light, agar-side down<br />
</ol><br />
<br />
<!-- Biobrick Assembly --><br />
<br><br />
<p class="title"><i>Biobrick Assembly</i></p><br />
<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p><br />
<ol><br />
<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone<br />
<li>To the same tube add 2.5 ul of NED buffer<br />
<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1<br />
<li>Incubate the tubes at 37℃ for 15 minutes<br />
<li>Then incubate at 80℃ for 20 minutes <br />
<li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone<br />
<li>Add 2 ul of 10X T4 DNA ligase restriction buffer<br />
<li>Add 1 ul of T4 DNA ligase <br />
<li>Incubate at room temperature for 10 minutes <br />
<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme<br />
</ol><br />
<p>This DNA can now be used for transformation</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
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<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title">References</h3><br />
<p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p><br />
<p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p><br />
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p><br />
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p><br />
<p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p><br />
<p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p><br />
</td><br />
</tr><br />
</table><br />
</html><br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/ProtocolsTeam:Reading/Protocols2014-10-18T00:53:26Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br />
<table><br />
<br />
<!--Protocols --><br />
<tr><br />
<td><h3 class="title">A note on protocols</h3></td><br />
</tr><br />
<br />
<!-- Introduction to the Protocols Page --><br />
<tr><br />
<td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p><br />
<br><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/0/0d/Oscar_doing_stuff.jpg" width="600"/></p><br />
</td><br />
<br />
<!-- Contents --><br />
<td width="27%" valign="top"><br />
<h3 class="title">Contents</h3><br />
<ol><br />
<li><a href="#">A Note on Protocols</a><br />
<li><a href="#protocols">Protocols</a><br />
<ul><br />
<li><a href="#prot1">Miniprep</a><br />
<li><a href="#prot2">Glycerol stock</a><br />
<li><a href="#prot3">Optical Density</a><br />
<li><a href="#prot4">PCR</a><br />
<li id=protocols><a href="#prot5">Transformation (E. coli)</a><br />
<li><a href="#prot6">Nanodrop</a><br />
<li id=prot1><a href="#prot7">BG-11 plates</a><br />
<li><a href="#prot8">Cyanobacteria Transformation</a><br />
<li><a href="#prot9">Biobrick Assembly</a></ul><br />
</ul><br />
<li><a href="#references">References</a><br />
<li><a href="#acknowledge">Acknowledgements</a><br />
</ol><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr><td colspan="3" height="15px"></td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"></tr><br />
<tr><td colspan="3" height="5px"></td></tr><br />
<br />
<!-- The Protocols --><br />
<tr><br />
<td colspan="3"><br />
<h3 class="title">Protocols</h3><br />
<br />
<!--Miniprep --><br />
<br><br />
<p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p><br />
<ol><br />
<li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes<br />
<li>Discard supernatant by pipetting off ensuring not to disturb the pellet<br />
<li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes<br />
<li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times<br />
<li>Centrifuge at 13,000 rpm for 5 minutes<br />
<li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate<br />
<li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm<br />
<li>Discard the flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li id=prot2>Discard flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Discard flow through<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube<br />
<li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette<br />
<li>Incubate at room temperature for 2 minutes<br />
<li>Centrifuge for 2 minutes at 13,000 rpm<br />
</ol><br />
<br />
<!-- Glycerol stock --><br />
<br><br />
<p class="title"><i>Making glycerol stock</i></p><br />
<p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p><br />
<ol><br />
<li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips<br />
<li>Centrifuge at 13,000 rpm for 2 minutes<br />
<li>Discard supernatant<br />
<li>Add 0.5 ml of 60% glycerol stock<br />
<ol style="list-style: lower-roman outside"><br />
<li>240 ml of glycerol<br />
<li>160 ml nano pure water<br />
<li>mix together and autoclave<br />
</ol><br />
<li>freeze at -80℃<br />
</ol><br />
<br />
<!-- Optical Density --><br />
<br><br />
<p class="title"><i>Taking optical density of culture</i></p><br />
<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p><br />
<ol><br />
<li>Set spectrophotometer to measure at OD<sub>750</sub><br />
<li>Blank with 1 ml of BG-11 in a cuvette<br />
<li>Measure 1 ml of culture<br />
<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11<br />
</ol><br />
<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p><br />
<br />
<!-- PCR --><br />
<br><br />
<p class="title"><i>PCR</i></p><br />
<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p><br />
<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p><br />
<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p><br />
<p>PCR program program as follows:</p><br />
<ol><br />
<li> Start:<br />
<ul><br />
<li>95℃ for 30 seconds<br />
</ul><br />
<li id=prot5>35 cycles:<br />
<ul><br />
<li>95℃ for 10 seconds<br />
<li>56℃ for 15 seconds<br />
<li>72℃ for 70 seconds<br />
</ul><br />
<li>Final<br />
<ul><br />
<li>72℃ for 5 minutes<br />
<li>68℃ for 10 minutes<br />
</ul><br />
</ol><br />
<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p><br />
<br />
<!-- Transformation (E. coli) --><br />
<br><br />
<p class="title"><i>Transformation into E. coli</i></p><br />
<p><br />
<ol><br />
<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf<br />
<li>Add 5 ul of DNA using a sterile pipette tip<br />
<li>Flick the tubes to mix and then store on ice for 30 minutes<br />
<li>Heat shock in a water bath at 42℃ for 1 minute<br />
<li>Incubate on ice for 5 minutes<br />
<li id=prot6>Add 450 ul of SOC (we used LB instead)<br />
<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm<br />
<li>Invert Eppendorfs containing the cells several times<br />
<li>Plate out and incubate overnight at 37℃<br />
</ol><br />
<p> Notes on transformation efficency</p><br />
<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). <a id=prot7 />We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p><br />
<br />
<!-- Nanodrop --><br />
<br><br />
<p class="title"><i>Nanodrop</i></p><br />
<p>Nanodrop is used to check concentration in DNA often from a miniprep</p><br />
<ol><br />
<li>Set the Nanodrop to measure DNA<br />
<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank<br />
<li>Add 2 ul of sample and measure. Measurement should be in ng/ul<br />
</ol><br />
<br />
<!-- BG-11 plates --><br />
<br><br />
<p class="title"><i>BG-11 plates</i></p><br />
<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p><br />
<ol><br />
<li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise<br />
<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml<br />
<li>Agar melted in steamer and kept at 55℃ until needed for pouring<br />
</ol><br />
<p> If making a kanamycin cap:</p><br />
<ol><br />
<li>0.6% agar w/v<br />
<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan<br />
<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it<br />
<li>Leave for >1 week for Kan selection to occur<br />
</ol><br />
<br />
<!-- Cyanobacteria Transformation --><br />
<br><br />
<p class="title"><i>Cyanobacteria Transformation</i></p><br />
<p>Protocol to transform DNA into cyanobacteria</p><br />
<ol><br />
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days<br />
<li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant<br />
<li>Repeat step 2<br />
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant<br />
<li>Add 200ml fresh BG-11<br />
<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)<br />
<li>Incubate for 24 under light on a shaker at ~100 rpm<br />
<li>Plate the full 200ml and leave for 1-2 days<br />
<li>Add 3-4ml kan 0.6% agar BG-11<br />
<li>Leave under light for >1 week<br />
</ol><br />
<p> After many attempts to transform with Cyanobacteria we refined this protocol<br />
<ol><br />
<li>Grow 6803 to ~0.435<br />
<li>Take 1 ml, re-wash with BG-11<br />
<li>Re-suspend in 90ml BG-11<br />
<li>Plasmid DNA at ~270ng/ul<br />
<li>add 8ul to each tube<br />
<li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes<br />
<li>transfer to warm room at 28ºC and leave overnight<br />
<li>plate on Kan50 and leave under light, agar-side down<br />
</ol><br />
<br />
<!-- Biobrick Assembly --><br />
<br><br />
<p class="title"><i>Biobrick Assembly</i></p><br />
<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p><br />
<ol><br />
<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone<br />
<li>To the same tube add 2.5 ul of NED buffer<br />
<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1<br />
<li>Incubate the tubes at 37℃ for 15 minutes<br />
<li>Then incubate at 80℃ for 20 minutes <br />
<li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone<br />
<li>Add 2 ul of 10X T4 DNA ligase restriction buffer<br />
<li>Add 1 ul of T4 DNA ligase <br />
<li>Incubate at room temperature for 10 minutes <br />
<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme<br />
</ol><br />
<p>This DNA can now be used for transformation</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title">References</h3><br />
<p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p><br />
<p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p><br />
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p><br />
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p><br />
<p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p><br />
<p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p><br />
</td><br />
</tr><br />
</table><br />
</html><br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/ProtocolsTeam:Reading/Protocols2014-10-18T00:52:16Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br />
<table><br />
<br />
<!--Protocols --><br />
<tr><br />
<td><h3 class="title">A note on protocols</h3></td><br />
<td> <h3 class="title">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction to the Protocols Page --><br />
<tr><br />
<td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p><br />
<br><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/0/0d/Oscar_doing_stuff.jpg" width="600"/></p><br />
</td><br />
<br />
<!-- Contents --><br />
<td width="27%" valign="top"><br />
<ol><br />
<li><a href="#">A Note on Protocols</a><br />
<li><a href="#protocols">Protocols</a><br />
<ul><br />
<li><a href="#prot1">Miniprep</a><br />
<li><a href="#prot2">Glycerol stock</a><br />
<li><a href="#prot3">Optical Density</a><br />
<li><a href="#prot4">PCR</a><br />
<li id=protocols><a href="#prot5">Transformation (E. coli)</a><br />
<li><a href="#prot6">Nanodrop</a><br />
<li id=prot1><a href="#prot7">BG-11 plates</a><br />
<li><a href="#prot8">Cyanobacteria Transformation</a><br />
<li><a href="#prot9">Biobrick Assembly</a></ul><br />
</ul><br />
<li><a href="#references">References</a><br />
<li><a href="#acknowledge">Acknowledgements</a><br />
</ol><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line --><br />
<tr><td colspan="3" height="15px"></td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"></tr><br />
<tr><td colspan="3" height="5px"></td></tr><br />
<br />
<!-- The Protocols --><br />
<tr><br />
<td colspan="3"><br />
<h3 class="title">Protocols</h3><br />
<br />
<!--Miniprep --><br />
<br><br />
<p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p><br />
<ol><br />
<li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes<br />
<li>Discard supernatant by pipetting off ensuring not to disturb the pellet<br />
<li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes<br />
<li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times<br />
<li>Centrifuge at 13,000 rpm for 5 minutes<br />
<li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate<br />
<li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm<br />
<li>Discard the flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li id=prot2>Discard flow through<br />
<li>Add 500 µl of wash solution to the column<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Discard flow through<br />
<li>Centrifuge for 1 minute at 13,000 rpm<br />
<li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube<br />
<li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette<br />
<li>Incubate at room temperature for 2 minutes<br />
<li>Centrifuge for 2 minutes at 13,000 rpm<br />
</ol><br />
<br />
<!-- Glycerol stock --><br />
<br><br />
<p class="title"><i>Making glycerol stock</i></p><br />
<p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p><br />
<ol><br />
<li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips<br />
<li>Centrifuge at 13,000 rpm for 2 minutes<br />
<li>Discard supernatant<br />
<li>Add 0.5 ml of 60% glycerol stock<br />
<ol style="list-style: lower-roman outside"><br />
<li>240 ml of glycerol<br />
<li>160 ml nano pure water<br />
<li>mix together and autoclave<br />
</ol><br />
<li>freeze at -80℃<br />
</ol><br />
<br />
<!-- Optical Density --><br />
<br><br />
<p class="title"><i>Taking optical density of culture</i></p><br />
<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p><br />
<ol><br />
<li>Set spectrophotometer to measure at OD<sub>750</sub><br />
<li>Blank with 1 ml of BG-11 in a cuvette<br />
<li>Measure 1 ml of culture<br />
<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11<br />
</ol><br />
<p>Converting OD<sub>750</sub> to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p><br />
<br />
<!-- PCR --><br />
<br><br />
<p class="title"><i>PCR</i></p><br />
<p>PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.</p><br />
<p> Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. </p><br />
<p>2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix <sup><a href="#references">6</a></sup></p><br />
<p>PCR program program as follows:</p><br />
<ol><br />
<li> Start:<br />
<ul><br />
<li>95℃ for 30 seconds<br />
</ul><br />
<li id=prot5>35 cycles:<br />
<ul><br />
<li>95℃ for 10 seconds<br />
<li>56℃ for 15 seconds<br />
<li>72℃ for 70 seconds<br />
</ul><br />
<li>Final<br />
<ul><br />
<li>72℃ for 5 minutes<br />
<li>68℃ for 10 minutes<br />
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<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p><br />
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<p class="title"><i>Transformation into E. coli</i></p><br />
<p><br />
<ol><br />
<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf<br />
<li>Add 5 ul of DNA using a sterile pipette tip<br />
<li>Flick the tubes to mix and then store on ice for 30 minutes<br />
<li>Heat shock in a water bath at 42℃ for 1 minute<br />
<li>Incubate on ice for 5 minutes<br />
<li id=prot6>Add 450 ul of SOC (we used LB instead)<br />
<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm<br />
<li>Invert Eppendorfs containing the cells several times<br />
<li>Plate out and incubate overnight at 37℃<br />
</ol><br />
<p> Notes on transformation efficency</p><br />
<p>Expected transformation efficiency is 1 x 10<sup>6</sup> cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). <a id=prot7 />We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed <sup><a href="#references">8</a></sup>.</p><br />
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<p class="title"><i>Nanodrop</i></p><br />
<p>Nanodrop is used to check concentration in DNA often from a miniprep</p><br />
<ol><br />
<li>Set the Nanodrop to measure DNA<br />
<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank<br />
<li>Add 2 ul of sample and measure. Measurement should be in ng/ul<br />
</ol><br />
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<p class="title"><i>BG-11 plates</i></p><br />
<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p><br />
<ol><br />
<li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise<br />
<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml<br />
<li>Agar melted in steamer and kept at 55℃ until needed for pouring<br />
</ol><br />
<p> If making a kanamycin cap:</p><br />
<ol><br />
<li>0.6% agar w/v<br />
<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan<br />
<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it<br />
<li>Leave for >1 week for Kan selection to occur<br />
</ol><br />
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<!-- Cyanobacteria Transformation --><br />
<br><br />
<p class="title"><i>Cyanobacteria Transformation</i></p><br />
<p>Protocol to transform DNA into cyanobacteria</p><br />
<ol><br />
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days<br />
<li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant<br />
<li>Repeat step 2<br />
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant<br />
<li>Add 200ml fresh BG-11<br />
<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)<br />
<li>Incubate for 24 under light on a shaker at ~100 rpm<br />
<li>Plate the full 200ml and leave for 1-2 days<br />
<li>Add 3-4ml kan 0.6% agar BG-11<br />
<li>Leave under light for >1 week<br />
</ol><br />
<p> After many attempts to transform with Cyanobacteria we refined this protocol<br />
<ol><br />
<li>Grow 6803 to ~0.435<br />
<li>Take 1 ml, re-wash with BG-11<br />
<li>Re-suspend in 90ml BG-11<br />
<li>Plasmid DNA at ~270ng/ul<br />
<li>add 8ul to each tube<br />
<li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes<br />
<li>transfer to warm room at 28ºC and leave overnight<br />
<li>plate on Kan50 and leave under light, agar-side down<br />
</ol><br />
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<!-- Biobrick Assembly --><br />
<br><br />
<p class="title"><i>Biobrick Assembly</i></p><br />
<p>Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:</p><br />
<ol><br />
<li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone<br />
<li>To the same tube add 2.5 ul of NED buffer<br />
<li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1<br />
<li>Incubate the tubes at 37℃ for 15 minutes<br />
<li>Then incubate at 80℃ for 20 minutes <br />
<li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone<br />
<li>Add 2 ul of 10X T4 DNA ligase restriction buffer<br />
<li>Add 1 ul of T4 DNA ligase <br />
<li>Incubate at room temperature for 10 minutes <br />
<li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme<br />
</ol><br />
<p>This DNA can now be used for transformation</p><br />
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<td colspan="3"><h3 class="title">References</h3><br />
<p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p><br />
<p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p><br />
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p><br />
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p><br />
<p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p><br />
<p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p><br />
<p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/File:Oscar_doing_stuff.jpgFile:Oscar doing stuff.jpg2014-10-18T00:49:28Z<p>Seafloor: uploaded a new version of &quot;File:Oscar doing stuff.jpg&quot;</p>
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<div></div>Seafloorhttp://2014.igem.org/Team:Reading/Human_PracticesTeam:Reading/Human Practices2014-10-18T00:42:14Z<p>Seafloor: </p>
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<h3 class="title" id="summary"> Regulatory challenges of rooftop installations</h3><br />
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<p><br />
An important part of iGEM is thinking about the wider impact of your project. We considered whether it would be possible to set up our cyanobacterial solar panels on roofs at Reading or on people’s houses. This meant coming up with a <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">design for a larger photovoltaic cell</a>, considering the biosafety issues involved, and what regulatory challenges we would face.<br />
</p><br />
<p><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/3/30/Cyano_cultures.jpg" width="800px"></p><br />
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<p><h3 class="title"> Contents</h3></p><br />
<p><br />
<ol><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#levels">Levels of Regulation</a></li><br />
<li><a href="#eu">EU Regulations</a></li><br />
<ul><br />
<li><a href="#euintro">Introduction</a></li><br />
<li><a href="#eucontained">Contained Use</a></li><br />
<li><a href="#eudelib">Deliberate Release</a></li><br />
</ul><br />
<li><a href="#uk">UK Regulations</a></li><br />
<ul><br />
<li><a href="#ukintro">Introduction</a></li><br />
<li><a href="#ukcontained">Contained Use</a></li><br />
</ul><br />
<li><a href="#other">Other Regulations</a></li><br />
<li><a href="#safety">Biosafety</a></li><br />
<li><a href="#conc">Findings and Conclusions</a></li><br />
<li><a href="#road">The Roadmap</a></li><br />
<li><a href="#resources">Resources</a></li><br />
<li><a href="#acknowledgements">Acknowledgements</a></li><br />
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<td colspan="3"><h3 class="title" id="intro">Introduction</h3><br />
<p><br />
Creating a cyanobacterial photovoltaic cell and getting it installed on a roof at a university presents a number of challenges. The design of the system is the first aspect to consider. We look into design and cost of the parts on the Fuel Cell page. Then there are European Union (EU) and government regulations and, in the case of Reading, several boards and internal committees through which applications would have to pass. We consider these in this section. Each of these raises questions about biosafety, such as potential effects of the escape of our organism into surrounding environments. In addition to using our technology at our own university, we also considered commercialising the technology and installing it on people’s houses. This opens up a new realm of issues, such as getting our energy source classed as renewable according to the EU’s Renewable Energy Directive (RED), and getting permission for having GMOs on many distinct properties. These wider problems are also reviewed here.<br />
</p><br />
<p><br />
Many sections of our report will be applicable to other teams considering contained use of GMOs, and we hope future teams will benefit from our research. The EU section will be particularly relevant to other EU member states, as the EU regulations form the common minimum requirements for each country. In general, this page should guide teams considering biosafety issues associated with cyanobacteria; there are currently no reviews of biosafety in synthetic biology of cyanobacteria that we are aware of. We finish with a roadmap for those thinking about whether they could commercialise a genetically modified microorganism (GMM)-containing system, especially as a renewable fuel source.<br />
</p><br />
<p><br />
It should be noted that the report mainly refers to contained use of GMMs. Though our system is contained, parts of it could be considered to overlap with deliberate release. We have therefore focussed on regulations pertaining to contained use, but have referred to those on deliberate release where our system could potentially fall under its purview. Due to this relevance, and partly time due to time constraints, we have not exhaustively considered deliberate release or contained use categorised as class 2 or above.<br />
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<td colspan="3"><h3 class="title" id="levels">The Different Levels of Regulations</h3><br />
<p><br />
At the highest level, the Cartagena Protocol on Biosafety covers living modified organisms (LMOs) and their transport across borders. This is an international United Nations agreement that has been in place since 2003, and is implemented in the EU by <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>. Below that, the EU issues “directives” on genetically modified organisms (GMOs) that must be implemented by all EU member states. For contained use, only the state’s regulations need to be considered; there is no involvement at the EU level. For deliberate release, rules are much more complicated, involving notification of the European Commision (EC), and will not be covered here. In the UK, the EU directives are implemented by the Department for Environment, Food and Rural Affairs (DEFRA) and the Health and Safety Executive (HSE). Finally, at Reading we would have to pass at least 3 committees - including the sub-committee for biological safety, the project committee, and the environmental committee - in addition to getting approval from the building manager.<br />
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<td colspan="3"><h3 class="title" id="eu">EU Regulations</h3><br />
<p class="title" id="euintro"><i>Introduction to EU Regulations</i></p><br />
<p><br />
The two EU directives concerning our plans are the directive 2009/41/EC on contained use and the 2001/18/EC directive on deliberate release of GMOs. Of these, the contained use directive is probably most appropriate. The 2009/41/EC directive defines contained use as: “any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment”, in Article 2(c).<br />
</p><br />
<p><br />
By contrast, the 2001/18/EC directive defines deliberate release as: “any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment”, in Article 2(3).<br />
</p><br />
<p><br />
By comparing these, and reviewing <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the proposed implementation of our idea</a>, we can see that we would most likely fall under the contained use directive because of our suggested containment measures. Our technology will use all of the activities specified by contained use, and implement appropriate safety measures. Based on this, we shall chiefly address contained use regulations, but mention rules on environmental release that are significant.<br />
</p><br />
<br /><br />
<p class="title" id="eucontained"><i>EU: Contained Use</i></p><br />
<p><br />
In summary, class 1 contained use requires: <br /><br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment</li><br />
<li>classification of the risk of the GMM according to the assessment</li><br />
<li>appropriate containment</li><br />
<li>notification of the relevant authority</li><br />
<li>an emergency plan for accidental release</li><br />
</ul><br />
</p><br />
<p><br />
We shall explore each of these points in further detail.<br />
</p><br />
<p><br />
Article 4 defines one of the main requirements for contained use: for a risk assessment to be carried out (Article 4(2)), in accordance with the guidelines in Annex III, with the aim of classifying the GMM. The criteria include assessing the potential to cause disease, effect on the environment (Annex III (A1)), and harmful effects of the genetic material, recipient, donor, vector and final GMM (Annex III (A2)). The severity of these issues and the chance of them happening must also be analysed. As a non-pathogenic organism, capability of causing disease is not relevant to our organism. The most germaine section is in Annex III (A1), which lists considering “deleterious effects due to establishment or dissemination in the environment” and “deleterious effects due to the natural transfer of inserted genetic material to other organisms.” Annex III (B7) goes on to require that the proposed use of the microorganism be combined with the above assessment in assigning it to a class. “Non-standard operations” is mentioned as affecting classification (Annex III (B7 iii)); this term is ambiguous, but may encompass our suggestion of having GMMs on roofs of private properties. <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">Our drip-in/drip-out system</a>, with the filter needing autoclaving upon replacement, may also fall under non-standard use. This would need to be taken into consideration if attempting to use our technology commercially. From this, it is clear that our GMM belongs in class 1 (as defined in Article 4(3)), so requires level 1 containment measures, though any doubt raised from our “non-standard operations” might cause a more strict classification (Article 4(4)). For those wishing to classify their organism, Directive 2000/54/EC can be referred to, or classification systems of the specific country.<br />
</p><br />
<p><br />
The risk assessment has a particular focus on waste disposal (Article 4(5)), making our drip-out waste disposal system very important. As removing the filter from the panel might be a source of accidental release, careful planning of waste management should be high on our priority list. The final risk assessment must be given to the competent authority (Article 4(6)) - HSE in the case of the UK. Containment measures are defined in Annex IV.<br />
</p><br />
<p><br />
Article 6 poses a potential issue. It requires notifying authorities upon contained use at each new property. While this is reasonable for single use at the university, our idea of having installations on separate houses would mean giving the information listed in Annex V for each site, including the risk assessment, which individuals are responsible for supervising, and a description of the premises. This would mean that the risk assessment must be sufficiently comprehensive to envision all potential environments where an installation may be set up, and would mean extra administration work for our organisation. It may be that, in the future, EU directives would need to be altered in order to make GMM technologies like ours more easily available for public benefit. Deliberate release regulations already contain a separate section for commercial use, and contained use may be separated this way in the future too.<br />
</p><br />
<p><br />
For class 1 organisms, no further notification is needed before commencing with contained use (Article 7). For higher risk classes more information is needed; as our organism is only class 1 we will not consider this, but rules can be found in Articles 8 and 9.<br />
</p><br />
<p><br />
Further thought should be given to the minimum containment measures stipulated in Annex IV, and whether our system meets these conditions. There are different requirements given for different potential situations. Our proposition would most likely fall under “Containment and other protective measures for other activities” for the panel itself, but other containment procedures would need to be reviewed for labs where genetic modification is done and areas where GMMs are cultured. Almost all containment options for class 1 organisms in this category are optional according the Annex IV. It is likely that the level of containment assumed for this category is more severe than in our proposition (i.e. the EC has assumed all contained use will occur inside a building). As such, we should expect to see some or all of the containment measures to be required, rather than optional, for our project. <br />
</p><br />
<p><br />
Beyond the obvious physical containment, there are several possible containment measures we could be required to enforce. This includes control of aerosols during “addition of material to a closed system or transfer of material to another system”. This would include transferring cyanobacteria to the fuel cell, which would be done in a separate contained facility, and during the removal of waste or the waste filter <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">(see design section)</a>, which would have to be done on site. The latter is one of the biggest issues our project could face.<br />
</p><br />
<p><br />
Inactivation of waste containing GMMs is also listed as optional, but could potentially be required. Furthermore, the air leaving the system, assuming filter-sterilised air is bubbled through our panel, could have to be filtered to prevent or minimise release. The only point already required for class 1 work is that personnel wear protective clothing.<br />
</p><br />
<p><br />
According to Article 13, an emergency plan is required. This must be made available to the public, relevant bodies and authorities, and other concerned EU member states. The plan is required in case containment measures fail, leading to “serious danger, whether immediate or delayed, to humans outside the premises and/or to the environment”. No information is given on how extensive this plan should be, and no minimum requirements are given. Member state legislation must therefore be consulted for any rules on how the plan must be structured.<br />
</p><br />
<p><br />
Finally, it should be noted that member states may consult the public on the proposition if they decide it is relevant (Article 12).<br />
</p><br />
<br /><br />
<p class="title" id="eudelib"><i>EU: Deliberate Release</i></p><br />
<p><br />
Below is outlined some of the salient points from the EU directive on deliberate release. These may be useful to other teams reviewing regulations. The key points are:<br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment is required (Article 4; Annex III)</li><br />
<li>regulations are different for commercial and non-commercial use</li><br />
<li>for non-commercial GMO work (Part B):</li><br />
<ul><br />
<li>parties must give a risk assessment and monitor use and effects</li><br />
<li>the authority can tell the public</li><br />
<li>approved uses must be reported to the EU</li><br />
</ul><br />
<li>for commercial GMO work (Part C):</li><br />
<ul><br />
<li>parties must notify the relevant authority before placing the product on the market</li><br />
<li>putting it on the market is defined as making it available to 3rd parties</li><br />
<li>the authority produces an “assessment report”</li><br />
<li>this is given to the applicant, the EC and EU member states</li><br />
<li>decisions apply throughout the EU</li><br />
<li>the public must be notified</li><br />
<li>as of June 2014, <a href="http://ec.europa.eu/food/plant/gmo/legislation/future_rules_en.htm">member states can restrict or ban GMOs in their country</a> that have been approved for all states</li><br />
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<p class="title" id="ukintro"><i>Introduction to UK Regulations</i></p><br />
<p><br />
<p><br />
The EU regulations are useful to us as they provide a baseline level of regulation we can expect if we try to implement our technology anywhere in the EU. In each EU member state, it is ultimately that country’s regulations which we must abide by. We will now consider what the regulations are like the UK. <br />
</p><br />
<p><br />
In general, they are slightly stricter than the basic EU regulations. The main regulations are <a href="http://www.hse.gov.uk/pubns/priced/l29.pdf">the HSE Contained Use regulations</a>, which are newly updated for 2014, and the accompanying SACGM Compendium of Guidance, which has yet to be updated to meet the new Contained Use document. Along with this, there are <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the regulations on deliberate release</a> from 1997, <a href="http://www.legislation.gov.uk/ukpga/1990/43/contents">section 108(1) of the Environment Protection Act </a> from 1990, and <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the Genetically Modified Organisms Regulations </a> (1996) that are related to GMOs. The latter three are all focussed on environmental release, so won’t be covered here. Regulations are upheld by the HSE and DEFRA. <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">The HSE website</a> can be consulted for all other regulations that might relate to the use of GMOs.<br />
</p><br />
<br /><br />
<p class="title" id="ukcontained"><i>UK Regulations: Contained Use</i></p><br />
<p><br />
The essential requirements for contained use in the UK are in the line with EU rules; we need to carry out a risk assessment and classify our organism, and notify the HSE before commencing GM work. So far we have only talked about the sites where the panels will be installed, but we will also have to consider regulations for handling, transport, work area decontamination, inactivation of GMMs and their disposal (including waste management). While the GMMs’ safety would need to be assessed by HSE, the system containing them, our panel, will need to be tested for leakages, with this evidence submitted to DEFRA.<br />
</p><br />
<p><br />
The definition given for contained use in Part 1, regulation 2 is “an activity in which organisms are genetically modified or in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers, or any combination of such barriers, are used to limit their contact with, and to provide a high level of protection for, humans and the environment”. Also in part 1, regulations 26 specifically tells us that commercial disposal of waste containing GMOs also falls under contained use. The contained use definition is similar to the EU definition, but is slightly more specific about what the containment measures must entail. In Part 1, paragraphs 24 and 45 give examples of what the barriers for our system might be expected to be. Physical could include a container, which would be the panel itself in our case. Chemical barriers may cover inactivation before waste disposal, and biological would include attenuating characteristics that debilitate the organism so that it is “rendered unable to survive outside of a specialised environment”. These barriers are discussed further in the safety page.<br />
</p><br />
<p><br />
The first requirement to consider is the risk assessment. For GMMs this is covered in Part 2, regulation 5, with more details on the assessment in Schedule 3 (Part 2). More emphasis is placed on risk to human and health and environment than in EU regulations. Regulation 5, paragraph 43, also answers questions we raised in the “EU: Contained Use” section about whether we could apply the same risk assessment to multiple sites with the same roof installation: “Where the contained use is identical at the multiple sites (eg in a clinical trial), the same risk assessment may apply to all the sites”. However it does point out that local changes in practices need to be taken into account. For us, practices would remain the same, but the surrounding environment may be different (e.g. there may be a pond at one property, where our organisms could theoretically survive). What the risk assessment must encompass is introduced in paragraph 44. In short, we must outline our plans, potential harmful effects, the chance of them occurring and their severity, and how we’ll deal with waste. These topics are covered in the safety page, where we look at each of our mutations; waste disposal is covered in <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the Fuel Cell page</a>.<br />
</p><br />
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<p><br />
It is clear that the detail needed in the risk assessment is partly defined by how well-understood the microorganism and mutations are. Although our organism is clearly a non-pathogenic, non-hazardous class 1 organism, it is not as well understood as Escherichia coli K-12, for example, and the genes are not ones that are commonly used. They do not have a strong history of safe use, like green fluorescent protein (GFP). All our mutations have been done before, however, while measuring for different endpoints, so literature is available for reference on the effects of our mutations. From paragraphs 52 and 53, we know that the classification of the work changes to reflect the level of containment needed. When considering EU regulations, we were unsure of the extent of containment required, as our organism is class 1, but is used in unusual and potentially problematic environments. Under UK regulations, it appears our work could be reclassified to class 2 if we deem the containment measures for class 2 work to be desirable. This brings in a previously unforeseen hurdle: our work may be relabelled as higher than class 1 because of the containment measures needed on private properties with no trained personnel. We will continue to assume our work is class 1, but mention class 2 rules where appropriate.<br />
</p><br />
<p><br />
In summary, for the risk assessment we must identify hazards, assign appropriate containment measures, then reclassify our work based on these (if necessary). These instructions are similar to, but more detailed than, those for EU member states in general.<br />
</p><br />
<p><br />
According to regulation 8, we need to obtain advice from a person or committee on the risk assessment. If our organism were reclassified as class 2, this would have to be a biological safety committee. At Reading this would not be an issue, as there is already a committee from which we could obtain advice. If the classification remained as class 1, our meeting with Gretta Roberts and Professor Jim Dunwell, who have advised us with regards to regulations and safety, should be sufficient.<br />
</p><br />
<p><br />
Regulation 9 requires notification of premises to be employed for contained use. We must first submit the information in Schedule 5, then wait 10 days for a response. A single notification may include more than 1 premises in situations where more than one premise is owned by the same company, so it’s possible that multiple sites could be asked about at once. This would reduce the cost of notifying HSE, which is currently <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£472 for class 1</a> and <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£943 for class 2</a>. However, if our organism still falls under class 1, we only need to submit a summary of the risk assessment, details on waste management and the advice we received during the risk assessment, and confirmation that relevant authorities will be notified of the emergency plan. The rest of the information needed is basic details like the address of the premises. For class 2 work, regulation 10 should be consulted.<br />
</p><br />
<p><br />
Part 3 outlines practices that must occur for contained use. Regulations 18, 19, 21 and 22 are all relevant to different stages of our plans. Sections relevant specifically to the panels include paragraphs 107-109, which tell us that containment measures must be tested; this may involve checking each panel for defects or leaking before deployment, and possibly visiting sites at intervals to check they are still functioning correctly (paragraph 108). This could mean simply looking over the panel for any cracks or leakages to check for physical containment. Checking that biological containment is still in place, for example by checking that cells cannot directly transfer or uptake DNA after the pilT1 mutation, may involve taking a sample from panels. This itself means removing liquid containing GMMs and transporting it back to the lab for testing transformation efficiency; we would have to ensure that taking any liquid samples would not have any risks of accidental release. It is unlikely, but possible, that we could be required to check for our GMM in the surrounding environment to ensure there had been no release (paragraph 111).<br />
</p><br />
<p><br />
Regulation 19 specifically covers containment measures for GMMs. Containment measures must be reviewed “at regular intervals” (paragraphs 128 and 129), which must occur more frequently for non-standard work. According to paragraph 134, our organism does not fit the criteria for class 1 work that does not require waste inactivation, because our organism does not contain “multiple disabling mutations”, so waste from our panel must be inactivated. According to 135, it is acceptable to take the filter to another location and autoclave it, assuming steps are taken to make sure storage and transport are safe, and that the process is effective at inactivating our GMM.<br />
</p><br />
<p><br />
We must follow Schedule 8, Part 2, Table 2 for containment measures. As with our assessment of EU containment requirements, we have chosen the “other” section, as this seems most appropriate. Here the only absolute requirement is that personnel must wear work clothing. Measures that might be required, if deemed so by the risk assessment, include: physical separation, control of aerosols (as discussed in the EU contained use section), inactivation of waste or removed fluid, and control for spillage. The last of these is the only one not fully considered so far in our design.<br />
</p><br />
<p><br />
Emergency plans are discussed in regulation 21. According to paragraph 139, however, “an emergency plan should only be prepared for work with organisms that pose the highest hazards to humans or the environment.” Although our organism is low hazard, the risk is perhaps higher because the GMMs are not contained in a facility. As only hazard is mentioned, it is possible that we would not have to draw up an emergency plan (for our organism the hazard is low, but the risk slightly higher as it is not contained in a building). Furthermore, only those on the premises would be exposed to our non-dangerous GMM, but paragraph 139 only requires an emergency plan when the health or safety of those outside the premises is in danger. This probably assumes the work is a designated building with trained personnel on site, though. Given this, and our non-standard plans, it would be prudent to anticipate an emergency plan being required. If this were the case, it must be submitted along with our contained used application to HSE (paragraph 140). Paragraph 141 covers the requirement for an emergency plan; those “on the site affected by the plan” should know the plan, so residents in houses where a panel is installed, or building managers for a university building, would have to be familiar with the plan (paragraph 142). The plan must also be publicly available (paragraph 143), as we know from the EU requirements. Although we have primarily talked about the risk to the environment of our GMMs, we should also consider the risks of releasing potassium ferricyanide to the environment (a part of our solar panel), like how much would be released, any risks to human health or safety, and any risks to the environment.<br />
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<p><br />
We’ve covered the main EU and UK regulations regarding the installation of our actual panels, but there are many other regulations relevant to our plans. Contained use regulations are the main ones, which would cover the genetic modification of our organisms in a lab, the transport of our organisms to a facility where they can be grown up, that facility itself, transport to houses or the university, and the property where the panel is installed. There are other regulations that could come into play, however. This includes rules on the <a href="http://www.hse.gov.uk/cdg/introduction.htm">carriage of dangerous goods</a>, which might include our GMMs or potassium ferricyanide. Furthermore, transport regulations for crossing borders in the EU, which might occur if we export cultivation of our GMMs to another country, would mean we must adhere to EU GMO border rules in <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>, which implements the Cartagena Protocol.<br />
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<p><br />
At Reading, we would also have to submit an application to at least 3 committees before getting our proposal approved, and speak to the building manager for each building we would want to get our panel put on. Although the committees sound like more regulatory hurdles, the university safety officers would contact HSE for us, and we would supply all our information to the safety officers, making the process much easier. Furthermore, the biological safety committee would be the committee we would contact for the expert advice required when carrying out a risk assessment, and all the buildings at the university count as one private property, for which only one application to HSE needs to be made.<br />
</p><br />
<p><br />
Commercialisation of our product would mean selling it as a new source of renewable energy. Renewable fuel sources are subject to other rules in the EU, which stipulate criteria that must be met for a source to be labelled as “renewable”. The Fuel Quality Directive (FQD) is relevant to renewable fuels for transport, such as biofuels, and the Renewable Energy Directive (RED) is pertinent to other energy sources that wish to be labelled as renewable; ours could fall under the latter. The requirements in RED are essentially requirements for EU member states, but in order for the standards to be met, it is individual companies that ultimately must comply. The RED is enforced by the European Commission Directorate General for Energy. The requirements include showing a reduction in greenhouse gas emissions over the course of the fuel’s production, and using life cycle analysis (LCA) methods to calculate the “carbon intensity” of our energy source. Our “sustainability analysis” must encompass other features beyond an LCA. To meet the specifications in the RED, we must check 12 independent factors of our energy source (including its production and transportation), and have this verified by a third party. The analysis method must be approved by the EU.<br />
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<p><br />
We have now reviewed the safety requirements at the EU and UK levels. The exact steps for the UK risk assessment are outlined in Section 3, part 2, of the Contained Use regulations. To meet the requirements for the UK, we must provide information on our organism. First, we will have to prove our organisms are less fit than the wild type. Under duress from changing lighting due to clouds, we think it likely that all our mutations will make our organisms less fit, so that they are outcompeted by the wild type. For HSE, we could show this by comparing growth rates of the two organisms under the same conditions, or comparing growth directly through a competition assay. In such a situation, we can assay for our mutant by PCRing for the BioBrick prefix and suffix (this is the same method we would use to identify our organism in the environment, if needed). Conditions for this should ideally be as realistic as possible, mimicking the temperature and lighting we would expect in the environments where our organism could be accidentally released.<br />
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Other evidence or information would need to be provided concerning DNA transfer. We would need to see which organisms our strain could potentially transfer genes to, and note the safety of those organisms (e.g. whether they are from a toxin-producing genus like <i>Microcystis</i> or <i>Anabaena</i>). As the modifications we are making are chromosomal, our modified DNA is much less mobilisable than plasmid DNA. Literature on genetic transfer in cyanobacteria is not as dense as it is for <i>Escherichia coli</i>, for example, so it is more difficult to be certain about mutation rates (which might be important in determining the chance of inactivating a kill switch) or which species DNA can be transferred to. As with all cells, lysing of our bacteria will release DNA into the environment. In our risk assessment, we should give evidence that any genes we introduce occur naturally (or could occur naturally). In our case, several of our mutations also carry kanamycin resistance. In this case, we would need to remove kanamycin resistance before using our organisms in an actual system. If we did leave in kanamycin resistance, we would again have to make it clear that kanamycin resistance genes occur naturally in the environment anyway. It should be noted that our organism does not produce toxins, and does not contribute to cyanobacterial blooms, so poses no obvious threat to the environment. It is also non-pathogenic so does not pose a threat to human health. It is possible that it could cause issues in an immunocompromised patient, though very unlikely. In our risk assessment, it would be best to look for cases of disease in immunocompromised patients being caused by <i>Synechocystis</i>, to show we are aware of potential issues.<br />
</p><br />
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<p><br />
For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.<br />
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<p><br />
Meeting experts in GMO safety and regulations, and consulting the appropriate legislation, has brought a number of key findings to light. Perhaps the most interesting is that current rules are not set up to cover a project like ours, that involves a container of GMMs outside and building, possibly on a person’s private property. As the Scientific Committees that in the EC are <a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">currently reviewing the risks of synthetic biology</a>, and that GMO-devices may become more common in the future, we may see regulations adapting more to cover these areas in the coming years.<br />
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<p><br />
Although there are clearly areas where our panel counts as non-standard use, and so might be reclassified as higher risk than class 1, passing regulations may not be our biggest hurdle in getting our technology to the market. Our system complies with the safety measures needed, and our organism is of no or negligible risk to human safety or health, or the environment. Other obstacles that might be difficult to pass include how heavy our panels will be, and whether this will be a problem for transport or installing on rooftops, as having employees or members of the public on roofs would be a large safety risk in itself. The cost of submitting contained use applications to HSE also needs to be taken into account, and how the panels will be maintained without trained personnel on site. Furthermore, aspects of scaling up our technology also need to be worked out, such as what the optimal ratio of cyanobacteria-inoculated media to potassium ferricyanide is, and whether our cyanobacteria will survive long-term use in a photovoltaic cell. In addition, we do not know if people will be interested in continuing to pay for new media that must be added. By contrast, normal photovoltaic cells are one-off payments, even if they would be much more expensive than a cyanobacterial photovoltaic cell.<br />
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<p><br />
This is a bullet-point guide of what we’d need to do, step-by-step, to get our product to the market. It also provides a summary of our findings. It is by no means exhaustive though. We have focussed on the panels, rather than other aspects of our hypothetical business, and expect that many hurdles would magically appear to make life more difficult if we attempted to carry out our proposal.<br />
</p><br />
<p><br />
<ul><br />
<li>Get proof that our organisms our less fit (competition assay)</li><br />
<li>Get proof that our system is secure (leaks)</li><br />
<li>Carry out a risk assessment</li><br />
<li>Get advice from an expert person or panel on the risk assessment</li><br />
<li>Provide appropriate containment measures</li><br />
<li>Review the class of our work in respect to the containment measures</li><br />
<li>Draw up an emergency plan, inform the relevant personnel.</li><br />
<li>Submit an application to HSE, pay £452 for each site</li><br />
<li>Wait 10 days for acknowledgement of receipt</li><br />
<li>Commence work</li><br />
</ul><br />
<p><br />
Before any of this, it would be advisable to contact HSE for advice on our proposition, however.<br />
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<p><br />
In this report we mainly referred to a few pieces of legislation, conversations we had with experts, and other sections of our wiki. As such, it made a lot more sense to link to all our resources as we went along, rather than using a Harvard or Vancouver style of referencing. However, we realise that it’s also convenient to have all the resources or references in one section. Here is a list of resources we used. If you’re hoping to review regulations on GMMs in the EU, this should be your starting point. <br />
</p><br />
<br /><br />
<p class="title"><i>Worldwide</i></p><br />
<p><br />
<b>The Cartagena Protocol</b> - UN-ratifed agreement for transborder GMO movement<br />
</p><br />
<br /><br />
<p class="title"><i>EU Directives</i></p><br />
<p><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009L0041&from=EN">Directive 2009/41/EC</a></b> - contained use <br /><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0018&from=EN">Directive 2001/18/EC</a></b> - deliberate release <br /><br />
<b>Directive 2000/54/EC</b> - risk classification of organisms <br /><br />
<b>Regulation EC 1946/2003</b> - transborder movement of GMOs. Implements the Cartagena Protocol <br /><br />
<b><a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">Opinion on Synthetic Biology</a></b> - first in a series to start reviewing risk in synthetic biology <br /><br />
For other EU legislation, start here <br /><br />
</p><br />
<br /><br />
<p class="title"><i>UK Regulations</i></p><br />
<p><br />
<b><a href="http://www.hse.gov.uk/pubns/books/l29.htm">Contained Use</a></b> <br /><br />
<b><a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/">SACGM Compendium of Guidance</a></b> <br /><br />
<b>Other UK rules</b> are mentioned <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">here</a> <br /><br />
<br />
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<br /><br />
<p class="title"><i>Other</i></p><br />
<p><br />
We also found the <a href="http://biofuelpolicywatch.wordpress.com">BioFuel Policy Watch</a> blog and <a href="http://dglassassociates.wordpress.com">its associated blog</a> to be useful for general information. David Glass’s blog post on <a href="http://dglassassociates.wordpress.com/2013/09/22/regulation-of-industrial-use-of-algae-or-cyanobacteria-in-europe-part-1/">EU regulations for algae and cyanobacteria</a> is also a great starting point.<br />
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<p><br />
When we first asked the question, “can we put our bacteria on a roof?”, we didn’t envision giving such a detailed response. The proposition only reached its current form through repeated rounds of meetings with the students and supervisors, time spent reading EU and UK regulations and, most importantly, meetings with experts in biosafety at Reading. Gretta Roberts and Professor Jim Dunwell were very kind in giving up their time to answer all our questions, and we are very grateful for their input.<br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Human_PracticesTeam:Reading/Human Practices2014-10-18T00:40:59Z<p>Seafloor: </p>
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An important part of iGEM is thinking about the wider impact of your project. We considered whether it would be possible to set up our cyanobacterial solar panels on roofs at Reading or on people’s houses. This meant coming up with a <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">design for a larger photovoltaic cell</a>, considering the biosafety issues involved, and what regulatory challenges we would face.<br />
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<li><a href="#summary">Summary</a></li><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#levels">Levels of Regulation</a></li><br />
<li><a href="#eu">EU Regulations</a></li><br />
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<li><a href="#euintro">Introduction</a></li><br />
<li><a href="#eucontained">Contained Use</a></li><br />
<li><a href="#eudelib">Deliberate Release</a></li><br />
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<li><a href="#uk">UK Regulations</a></li><br />
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<li><a href="#ukintro">Introduction</a></li><br />
<li><a href="#ukcontained">Contained Use</a></li><br />
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<li><a href="#other">Other Regulations</a></li><br />
<li><a href="#safety">Biosafety</a></li><br />
<li><a href="#conc">Findings and Conclusions</a></li><br />
<li><a href="#road">The Roadmap</a></li><br />
<li><a href="#resources">Resources</a></li><br />
<li><a href="#acknowledgements">Acknowledgements</a></li><br />
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<p><br />
Creating a cyanobacterial photovoltaic cell and getting it installed on a roof at a university presents a number of challenges. The design of the system is the first aspect to consider. We look into design and cost of the parts on the Fuel Cell page. Then there are European Union (EU) and government regulations and, in the case of Reading, several boards and internal committees through which applications would have to pass. We consider these in this section. Each of these raises questions about biosafety, such as potential effects of the escape of our organism into surrounding environments. In addition to using our technology at our own university, we also considered commercialising the technology and installing it on people’s houses. This opens up a new realm of issues, such as getting our energy source classed as renewable according to the EU’s Renewable Energy Directive (RED), and getting permission for having GMOs on many distinct properties. These wider problems are also reviewed here.<br />
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<p><br />
Many sections of our report will be applicable to other teams considering contained use of GMOs, and we hope future teams will benefit from our research. The EU section will be particularly relevant to other EU member states, as the EU regulations form the common minimum requirements for each country. In general, this page should guide teams considering biosafety issues associated with cyanobacteria; there are currently no reviews of biosafety in synthetic biology of cyanobacteria that we are aware of. We finish with a roadmap for those thinking about whether they could commercialise a genetically modified microorganism (GMM)-containing system, especially as a renewable fuel source.<br />
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<p><br />
It should be noted that the report mainly refers to contained use of GMMs. Though our system is contained, parts of it could be considered to overlap with deliberate release. We have therefore focussed on regulations pertaining to contained use, but have referred to those on deliberate release where our system could potentially fall under its purview. Due to this relevance, and partly time due to time constraints, we have not exhaustively considered deliberate release or contained use categorised as class 2 or above.<br />
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<td colspan="3"><h3 class="title" id="levels">The Different Levels of Regulations</h3><br />
<p><br />
At the highest level, the Cartagena Protocol on Biosafety covers living modified organisms (LMOs) and their transport across borders. This is an international United Nations agreement that has been in place since 2003, and is implemented in the EU by <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>. Below that, the EU issues “directives” on genetically modified organisms (GMOs) that must be implemented by all EU member states. For contained use, only the state’s regulations need to be considered; there is no involvement at the EU level. For deliberate release, rules are much more complicated, involving notification of the European Commision (EC), and will not be covered here. In the UK, the EU directives are implemented by the Department for Environment, Food and Rural Affairs (DEFRA) and the Health and Safety Executive (HSE). Finally, at Reading we would have to pass at least 3 committees - including the sub-committee for biological safety, the project committee, and the environmental committee - in addition to getting approval from the building manager.<br />
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<td colspan="3"><h3 class="title" id="eu">EU Regulations</h3><br />
<p class="title" id="euintro"><i>Introduction to EU Regulations</i></p><br />
<p><br />
The two EU directives concerning our plans are the directive 2009/41/EC on contained use and the 2001/18/EC directive on deliberate release of GMOs. Of these, the contained use directive is probably most appropriate. The 2009/41/EC directive defines contained use as: “any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment”, in Article 2(c).<br />
</p><br />
<p><br />
By contrast, the 2001/18/EC directive defines deliberate release as: “any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment”, in Article 2(3).<br />
</p><br />
<p><br />
By comparing these, and reviewing <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the proposed implementation of our idea</a>, we can see that we would most likely fall under the contained use directive because of our suggested containment measures. Our technology will use all of the activities specified by contained use, and implement appropriate safety measures. Based on this, we shall chiefly address contained use regulations, but mention rules on environmental release that are significant.<br />
</p><br />
<br /><br />
<p class="title" id="eucontained"><i>EU: Contained Use</i></p><br />
<p><br />
In summary, class 1 contained use requires: <br /><br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment</li><br />
<li>classification of the risk of the GMM according to the assessment</li><br />
<li>appropriate containment</li><br />
<li>notification of the relevant authority</li><br />
<li>an emergency plan for accidental release</li><br />
</ul><br />
</p><br />
<p><br />
We shall explore each of these points in further detail.<br />
</p><br />
<p><br />
Article 4 defines one of the main requirements for contained use: for a risk assessment to be carried out (Article 4(2)), in accordance with the guidelines in Annex III, with the aim of classifying the GMM. The criteria include assessing the potential to cause disease, effect on the environment (Annex III (A1)), and harmful effects of the genetic material, recipient, donor, vector and final GMM (Annex III (A2)). The severity of these issues and the chance of them happening must also be analysed. As a non-pathogenic organism, capability of causing disease is not relevant to our organism. The most germaine section is in Annex III (A1), which lists considering “deleterious effects due to establishment or dissemination in the environment” and “deleterious effects due to the natural transfer of inserted genetic material to other organisms.” Annex III (B7) goes on to require that the proposed use of the microorganism be combined with the above assessment in assigning it to a class. “Non-standard operations” is mentioned as affecting classification (Annex III (B7 iii)); this term is ambiguous, but may encompass our suggestion of having GMMs on roofs of private properties. <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">Our drip-in/drip-out system</a>, with the filter needing autoclaving upon replacement, may also fall under non-standard use. This would need to be taken into consideration if attempting to use our technology commercially. From this, it is clear that our GMM belongs in class 1 (as defined in Article 4(3)), so requires level 1 containment measures, though any doubt raised from our “non-standard operations” might cause a more strict classification (Article 4(4)). For those wishing to classify their organism, Directive 2000/54/EC can be referred to, or classification systems of the specific country.<br />
</p><br />
<p><br />
The risk assessment has a particular focus on waste disposal (Article 4(5)), making our drip-out waste disposal system very important. As removing the filter from the panel might be a source of accidental release, careful planning of waste management should be high on our priority list. The final risk assessment must be given to the competent authority (Article 4(6)) - HSE in the case of the UK. Containment measures are defined in Annex IV.<br />
</p><br />
<p><br />
Article 6 poses a potential issue. It requires notifying authorities upon contained use at each new property. While this is reasonable for single use at the university, our idea of having installations on separate houses would mean giving the information listed in Annex V for each site, including the risk assessment, which individuals are responsible for supervising, and a description of the premises. This would mean that the risk assessment must be sufficiently comprehensive to envision all potential environments where an installation may be set up, and would mean extra administration work for our organisation. It may be that, in the future, EU directives would need to be altered in order to make GMM technologies like ours more easily available for public benefit. Deliberate release regulations already contain a separate section for commercial use, and contained use may be separated this way in the future too.<br />
</p><br />
<p><br />
For class 1 organisms, no further notification is needed before commencing with contained use (Article 7). For higher risk classes more information is needed; as our organism is only class 1 we will not consider this, but rules can be found in Articles 8 and 9.<br />
</p><br />
<p><br />
Further thought should be given to the minimum containment measures stipulated in Annex IV, and whether our system meets these conditions. There are different requirements given for different potential situations. Our proposition would most likely fall under “Containment and other protective measures for other activities” for the panel itself, but other containment procedures would need to be reviewed for labs where genetic modification is done and areas where GMMs are cultured. Almost all containment options for class 1 organisms in this category are optional according the Annex IV. It is likely that the level of containment assumed for this category is more severe than in our proposition (i.e. the EC has assumed all contained use will occur inside a building). As such, we should expect to see some or all of the containment measures to be required, rather than optional, for our project. <br />
</p><br />
<p><br />
Beyond the obvious physical containment, there are several possible containment measures we could be required to enforce. This includes control of aerosols during “addition of material to a closed system or transfer of material to another system”. This would include transferring cyanobacteria to the fuel cell, which would be done in a separate contained facility, and during the removal of waste or the waste filter <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">(see design section)</a>, which would have to be done on site. The latter is one of the biggest issues our project could face.<br />
</p><br />
<p><br />
Inactivation of waste containing GMMs is also listed as optional, but could potentially be required. Furthermore, the air leaving the system, assuming filter-sterilised air is bubbled through our panel, could have to be filtered to prevent or minimise release. The only point already required for class 1 work is that personnel wear protective clothing.<br />
</p><br />
<p><br />
According to Article 13, an emergency plan is required. This must be made available to the public, relevant bodies and authorities, and other concerned EU member states. The plan is required in case containment measures fail, leading to “serious danger, whether immediate or delayed, to humans outside the premises and/or to the environment”. No information is given on how extensive this plan should be, and no minimum requirements are given. Member state legislation must therefore be consulted for any rules on how the plan must be structured.<br />
</p><br />
<p><br />
Finally, it should be noted that member states may consult the public on the proposition if they decide it is relevant (Article 12).<br />
</p><br />
<br /><br />
<p class="title" id="eudelib"><i>EU: Deliberate Release</i></p><br />
<p><br />
Below is outlined some of the salient points from the EU directive on deliberate release. These may be useful to other teams reviewing regulations. The key points are:<br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment is required (Article 4; Annex III)</li><br />
<li>regulations are different for commercial and non-commercial use</li><br />
<li>for non-commercial GMO work (Part B):</li><br />
<ul><br />
<li>parties must give a risk assessment and monitor use and effects</li><br />
<li>the authority can tell the public</li><br />
<li>approved uses must be reported to the EU</li><br />
</ul><br />
<li>for commercial GMO work (Part C):</li><br />
<ul><br />
<li>parties must notify the relevant authority before placing the product on the market</li><br />
<li>putting it on the market is defined as making it available to 3rd parties</li><br />
<li>the authority produces an “assessment report”</li><br />
<li>this is given to the applicant, the EC and EU member states</li><br />
<li>decisions apply throughout the EU</li><br />
<li>the public must be notified</li><br />
<li>as of June 2014, <a href="http://ec.europa.eu/food/plant/gmo/legislation/future_rules_en.htm">member states can restrict or ban GMOs in their country</a> that have been approved for all states</li><br />
</ul><br />
</ul><br />
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<td colspan="3"><h3 class="title" id="uk">UK Regulations</h3><br />
<p class="title" id="ukintro"><i>Introduction to UK Regulations</i></p><br />
<p><br />
<p><br />
The EU regulations are useful to us as they provide a baseline level of regulation we can expect if we try to implement our technology anywhere in the EU. In each EU member state, it is ultimately that country’s regulations which we must abide by. We will now consider what the regulations are like the UK. <br />
</p><br />
<p><br />
In general, they are slightly stricter than the basic EU regulations. The main regulations are <a href="http://www.hse.gov.uk/pubns/priced/l29.pdf">the HSE Contained Use regulations</a>, which are newly updated for 2014, and the accompanying SACGM Compendium of Guidance, which has yet to be updated to meet the new Contained Use document. Along with this, there are <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the regulations on deliberate release</a> from 1997, <a href="http://www.legislation.gov.uk/ukpga/1990/43/contents">section 108(1) of the Environment Protection Act </a> from 1990, and <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the Genetically Modified Organisms Regulations </a> (1996) that are related to GMOs. The latter three are all focussed on environmental release, so won’t be covered here. Regulations are upheld by the HSE and DEFRA. <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">The HSE website</a> can be consulted for all other regulations that might relate to the use of GMOs.<br />
</p><br />
<br /><br />
<p class="title" id="ukcontained"><i>UK Regulations: Contained Use</i></p><br />
<p><br />
The essential requirements for contained use in the UK are in the line with EU rules; we need to carry out a risk assessment and classify our organism, and notify the HSE before commencing GM work. So far we have only talked about the sites where the panels will be installed, but we will also have to consider regulations for handling, transport, work area decontamination, inactivation of GMMs and their disposal (including waste management). While the GMMs’ safety would need to be assessed by HSE, the system containing them, our panel, will need to be tested for leakages, with this evidence submitted to DEFRA.<br />
</p><br />
<p><br />
The definition given for contained use in Part 1, regulation 2 is “an activity in which organisms are genetically modified or in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers, or any combination of such barriers, are used to limit their contact with, and to provide a high level of protection for, humans and the environment”. Also in part 1, regulations 26 specifically tells us that commercial disposal of waste containing GMOs also falls under contained use. The contained use definition is similar to the EU definition, but is slightly more specific about what the containment measures must entail. In Part 1, paragraphs 24 and 45 give examples of what the barriers for our system might be expected to be. Physical could include a container, which would be the panel itself in our case. Chemical barriers may cover inactivation before waste disposal, and biological would include attenuating characteristics that debilitate the organism so that it is “rendered unable to survive outside of a specialised environment”. These barriers are discussed further in the safety page.<br />
</p><br />
<p><br />
The first requirement to consider is the risk assessment. For GMMs this is covered in Part 2, regulation 5, with more details on the assessment in Schedule 3 (Part 2). More emphasis is placed on risk to human and health and environment than in EU regulations. Regulation 5, paragraph 43, also answers questions we raised in the “EU: Contained Use” section about whether we could apply the same risk assessment to multiple sites with the same roof installation: “Where the contained use is identical at the multiple sites (eg in a clinical trial), the same risk assessment may apply to all the sites”. However it does point out that local changes in practices need to be taken into account. For us, practices would remain the same, but the surrounding environment may be different (e.g. there may be a pond at one property, where our organisms could theoretically survive). What the risk assessment must encompass is introduced in paragraph 44. In short, we must outline our plans, potential harmful effects, the chance of them occurring and their severity, and how we’ll deal with waste. These topics are covered in the safety page, where we look at each of our mutations; waste disposal is covered in <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the Fuel Cell page</a>.<br />
</p><br />
<br /><br />
<p><center><img align=centre src="https://static.igem.org/mediawiki/2014/7/77/Containment_lab_1.jpg" width="800px"></center></p><br />
<br /><br />
<p><br />
It is clear that the detail needed in the risk assessment is partly defined by how well-understood the microorganism and mutations are. Although our organism is clearly a non-pathogenic, non-hazardous class 1 organism, it is not as well understood as Escherichia coli K-12, for example, and the genes are not ones that are commonly used. They do not have a strong history of safe use, like green fluorescent protein (GFP). All our mutations have been done before, however, while measuring for different endpoints, so literature is available for reference on the effects of our mutations. From paragraphs 52 and 53, we know that the classification of the work changes to reflect the level of containment needed. When considering EU regulations, we were unsure of the extent of containment required, as our organism is class 1, but is used in unusual and potentially problematic environments. Under UK regulations, it appears our work could be reclassified to class 2 if we deem the containment measures for class 2 work to be desirable. This brings in a previously unforeseen hurdle: our work may be relabelled as higher than class 1 because of the containment measures needed on private properties with no trained personnel. We will continue to assume our work is class 1, but mention class 2 rules where appropriate.<br />
</p><br />
<p><br />
In summary, for the risk assessment we must identify hazards, assign appropriate containment measures, then reclassify our work based on these (if necessary). These instructions are similar to, but more detailed than, those for EU member states in general.<br />
</p><br />
<p><br />
According to regulation 8, we need to obtain advice from a person or committee on the risk assessment. If our organism were reclassified as class 2, this would have to be a biological safety committee. At Reading this would not be an issue, as there is already a committee from which we could obtain advice. If the classification remained as class 1, our meeting with Gretta Roberts and Professor Jim Dunwell, who have advised us with regards to regulations and safety, should be sufficient.<br />
</p><br />
<p><br />
Regulation 9 requires notification of premises to be employed for contained use. We must first submit the information in Schedule 5, then wait 10 days for a response. A single notification may include more than 1 premises in situations where more than one premise is owned by the same company, so it’s possible that multiple sites could be asked about at once. This would reduce the cost of notifying HSE, which is currently <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£472 for class 1</a> and <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£943 for class 2</a>. However, if our organism still falls under class 1, we only need to submit a summary of the risk assessment, details on waste management and the advice we received during the risk assessment, and confirmation that relevant authorities will be notified of the emergency plan. The rest of the information needed is basic details like the address of the premises. For class 2 work, regulation 10 should be consulted.<br />
</p><br />
<p><br />
Part 3 outlines practices that must occur for contained use. Regulations 18, 19, 21 and 22 are all relevant to different stages of our plans. Sections relevant specifically to the panels include paragraphs 107-109, which tell us that containment measures must be tested; this may involve checking each panel for defects or leaking before deployment, and possibly visiting sites at intervals to check they are still functioning correctly (paragraph 108). This could mean simply looking over the panel for any cracks or leakages to check for physical containment. Checking that biological containment is still in place, for example by checking that cells cannot directly transfer or uptake DNA after the pilT1 mutation, may involve taking a sample from panels. This itself means removing liquid containing GMMs and transporting it back to the lab for testing transformation efficiency; we would have to ensure that taking any liquid samples would not have any risks of accidental release. It is unlikely, but possible, that we could be required to check for our GMM in the surrounding environment to ensure there had been no release (paragraph 111).<br />
</p><br />
<p><br />
Regulation 19 specifically covers containment measures for GMMs. Containment measures must be reviewed “at regular intervals” (paragraphs 128 and 129), which must occur more frequently for non-standard work. According to paragraph 134, our organism does not fit the criteria for class 1 work that does not require waste inactivation, because our organism does not contain “multiple disabling mutations”, so waste from our panel must be inactivated. According to 135, it is acceptable to take the filter to another location and autoclave it, assuming steps are taken to make sure storage and transport are safe, and that the process is effective at inactivating our GMM.<br />
</p><br />
<p><br />
We must follow Schedule 8, Part 2, Table 2 for containment measures. As with our assessment of EU containment requirements, we have chosen the “other” section, as this seems most appropriate. Here the only absolute requirement is that personnel must wear work clothing. Measures that might be required, if deemed so by the risk assessment, include: physical separation, control of aerosols (as discussed in the EU contained use section), inactivation of waste or removed fluid, and control for spillage. The last of these is the only one not fully considered so far in our design.<br />
</p><br />
<p><br />
Emergency plans are discussed in regulation 21. According to paragraph 139, however, “an emergency plan should only be prepared for work with organisms that pose the highest hazards to humans or the environment.” Although our organism is low hazard, the risk is perhaps higher because the GMMs are not contained in a facility. As only hazard is mentioned, it is possible that we would not have to draw up an emergency plan (for our organism the hazard is low, but the risk slightly higher as it is not contained in a building). Furthermore, only those on the premises would be exposed to our non-dangerous GMM, but paragraph 139 only requires an emergency plan when the health or safety of those outside the premises is in danger. This probably assumes the work is a designated building with trained personnel on site, though. Given this, and our non-standard plans, it would be prudent to anticipate an emergency plan being required. If this were the case, it must be submitted along with our contained used application to HSE (paragraph 140). Paragraph 141 covers the requirement for an emergency plan; those “on the site affected by the plan” should know the plan, so residents in houses where a panel is installed, or building managers for a university building, would have to be familiar with the plan (paragraph 142). The plan must also be publicly available (paragraph 143), as we know from the EU requirements. Although we have primarily talked about the risk to the environment of our GMMs, we should also consider the risks of releasing potassium ferricyanide to the environment (a part of our solar panel), like how much would be released, any risks to human health or safety, and any risks to the environment.<br />
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<td colspan="3"><h3 class="title" id="other">Other Regulations</h3><br />
<p><br />
We’ve covered the main EU and UK regulations regarding the installation of our actual panels, but there are many other regulations relevant to our plans. Contained use regulations are the main ones, which would cover the genetic modification of our organisms in a lab, the transport of our organisms to a facility where they can be grown up, that facility itself, transport to houses or the university, and the property where the panel is installed. There are other regulations that could come into play, however. This includes rules on the <a href="http://www.hse.gov.uk/cdg/introduction.htm">carriage of dangerous goods</a>, which might include our GMMs or potassium ferricyanide. Furthermore, transport regulations for crossing borders in the EU, which might occur if we export cultivation of our GMMs to another country, would mean we must adhere to EU GMO border rules in <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>, which implements the Cartagena Protocol.<br />
</p><br />
<p><br />
At Reading, we would also have to submit an application to at least 3 committees before getting our proposal approved, and speak to the building manager for each building we would want to get our panel put on. Although the committees sound like more regulatory hurdles, the university safety officers would contact HSE for us, and we would supply all our information to the safety officers, making the process much easier. Furthermore, the biological safety committee would be the committee we would contact for the expert advice required when carrying out a risk assessment, and all the buildings at the university count as one private property, for which only one application to HSE needs to be made.<br />
</p><br />
<p><br />
Commercialisation of our product would mean selling it as a new source of renewable energy. Renewable fuel sources are subject to other rules in the EU, which stipulate criteria that must be met for a source to be labelled as “renewable”. The Fuel Quality Directive (FQD) is relevant to renewable fuels for transport, such as biofuels, and the Renewable Energy Directive (RED) is pertinent to other energy sources that wish to be labelled as renewable; ours could fall under the latter. The requirements in RED are essentially requirements for EU member states, but in order for the standards to be met, it is individual companies that ultimately must comply. The RED is enforced by the European Commission Directorate General for Energy. The requirements include showing a reduction in greenhouse gas emissions over the course of the fuel’s production, and using life cycle analysis (LCA) methods to calculate the “carbon intensity” of our energy source. Our “sustainability analysis” must encompass other features beyond an LCA. To meet the specifications in the RED, we must check 12 independent factors of our energy source (including its production and transportation), and have this verified by a third party. The analysis method must be approved by the EU.<br />
</p><br />
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<td colspan="3"><h3 class="title" id="safety">Biosafety</h3><br />
<p><br />
We have now reviewed the safety requirements at the EU and UK levels. The exact steps for the UK risk assessment are outlined in Section 3, part 2, of the Contained Use regulations. To meet the requirements for the UK, we must provide information on our organism. First, we will have to prove our organisms are less fit than the wild type. Under duress from changing lighting due to clouds, we think it likely that all our mutations will make our organisms less fit, so that they are outcompeted by the wild type. For HSE, we could show this by comparing growth rates of the two organisms under the same conditions, or comparing growth directly through a competition assay. In such a situation, we can assay for our mutant by PCRing for the BioBrick prefix and suffix (this is the same method we would use to identify our organism in the environment, if needed). Conditions for this should ideally be as realistic as possible, mimicking the temperature and lighting we would expect in the environments where our organism could be accidentally released.<br />
</p><br />
<p><br />
Other evidence or information would need to be provided concerning DNA transfer. We would need to see which organisms our strain could potentially transfer genes to, and note the safety of those organisms (e.g. whether they are from a toxin-producing genus like <i>Microcystis</i> or <i>Anabaena</i>). As the modifications we are making are chromosomal, our modified DNA is much less mobilisable than plasmid DNA. Literature on genetic transfer in cyanobacteria is not as dense as it is for <i>Escherichia coli</i>, for example, so it is more difficult to be certain about mutation rates (which might be important in determining the chance of inactivating a kill switch) or which species DNA can be transferred to. As with all cells, lysing of our bacteria will release DNA into the environment. In our risk assessment, we should give evidence that any genes we introduce occur naturally (or could occur naturally). In our case, several of our mutations also carry kanamycin resistance. In this case, we would need to remove kanamycin resistance before using our organisms in an actual system. If we did leave in kanamycin resistance, we would again have to make it clear that kanamycin resistance genes occur naturally in the environment anyway. It should be noted that our organism does not produce toxins, and does not contribute to cyanobacterial blooms, so poses no obvious threat to the environment. It is also non-pathogenic so does not pose a threat to human health. It is possible that it could cause issues in an immunocompromised patient, though very unlikely. In our risk assessment, it would be best to look for cases of disease in immunocompromised patients being caused by <i>Synechocystis</i>, to show we are aware of potential issues.<br />
</p><br />
<br /><br />
<p><br />
For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.<br />
</p><br />
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<td colspan="3"><h3 class="title" id="conc">Findings and Conclusions</h3><br />
<p><br />
Meeting experts in GMO safety and regulations, and consulting the appropriate legislation, has brought a number of key findings to light. Perhaps the most interesting is that current rules are not set up to cover a project like ours, that involves a container of GMMs outside and building, possibly on a person’s private property. As the Scientific Committees that in the EC are <a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">currently reviewing the risks of synthetic biology</a>, and that GMO-devices may become more common in the future, we may see regulations adapting more to cover these areas in the coming years.<br />
</p><br />
<p><br />
Although there are clearly areas where our panel counts as non-standard use, and so might be reclassified as higher risk than class 1, passing regulations may not be our biggest hurdle in getting our technology to the market. Our system complies with the safety measures needed, and our organism is of no or negligible risk to human safety or health, or the environment. Other obstacles that might be difficult to pass include how heavy our panels will be, and whether this will be a problem for transport or installing on rooftops, as having employees or members of the public on roofs would be a large safety risk in itself. The cost of submitting contained use applications to HSE also needs to be taken into account, and how the panels will be maintained without trained personnel on site. Furthermore, aspects of scaling up our technology also need to be worked out, such as what the optimal ratio of cyanobacteria-inoculated media to potassium ferricyanide is, and whether our cyanobacteria will survive long-term use in a photovoltaic cell. In addition, we do not know if people will be interested in continuing to pay for new media that must be added. By contrast, normal photovoltaic cells are one-off payments, even if they would be much more expensive than a cyanobacterial photovoltaic cell.<br />
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<td colspan="3"><h3 class="title" id="road">The Roadmap</h3><br />
<p><br />
This is a bullet-point guide of what we’d need to do, step-by-step, to get our product to the market. It also provides a summary of our findings. It is by no means exhaustive though. We have focussed on the panels, rather than other aspects of our hypothetical business, and expect that many hurdles would magically appear to make life more difficult if we attempted to carry out our proposal.<br />
</p><br />
<p><br />
<ul><br />
<li>Get proof that our organisms our less fit (competition assay)</li><br />
<li>Get proof that our system is secure (leaks)</li><br />
<li>Carry out a risk assessment</li><br />
<li>Get advice from an expert person or panel on the risk assessment</li><br />
<li>Provide appropriate containment measures</li><br />
<li>Review the class of our work in respect to the containment measures</li><br />
<li>Draw up an emergency plan, inform the relevant personnel.</li><br />
<li>Submit an application to HSE, pay £452 for each site</li><br />
<li>Wait 10 days for acknowledgement of receipt</li><br />
<li>Commence work</li><br />
</ul><br />
<p><br />
Before any of this, it would be advisable to contact HSE to for advice on our proposition, however.<br />
</p><br />
</p><br />
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<td colspan="3"><h3 class="title" id="resources">Resources</h3><br />
<p><br />
In this report we mainly referred to a few pieces of legislation, conversations we had with experts, and other sections of our wiki. As such, it made a lot more sense to link to all our resources as we went along, rather than using a Harvard or Vancouver style of referencing. However, we realise that it’s also convenient to have all the resources or references in one section. Here is a list of resources we used. If you’re hoping to review regulations on GMMs in the EU, this should be your starting point. <br />
</p><br />
<br /><br />
<p class="title"><i>Worldwide</i></p><br />
<p><br />
<b>The Cartagena Protocol</b> - UN-ratifed agreement for transborder GMO movement<br />
</p><br />
<br /><br />
<p class="title"><i>EU Directives</i></p><br />
<p><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009L0041&from=EN">Directive 2009/41/EC</a></b> - contained use <br /><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0018&from=EN">Directive 2001/18/EC</a></b> - deliberate release <br /><br />
<b>Directive 2000/54/EC</b> - risk classification of organisms <br /><br />
<b>Regulation EC 1946/2003</b> - transborder movement of GMOs. Implements the Cartagena Protocol <br /><br />
<b><a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">Opinion on Synthetic Biology</a></b> - first in a series to start reviewing risk in synthetic biology <br /><br />
For other EU legislation, start here <br /><br />
</p><br />
<br /><br />
<p class="title"><i>UK Regulations</i></p><br />
<p><br />
<b><a href="http://www.hse.gov.uk/pubns/books/l29.htm">Contained Use</a></b> <br /><br />
<b><a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/">SACGM Compendium of Guidance</a></b> <br /><br />
<b>Other UK rules</b> are mentioned <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">here</a> <br /><br />
<br />
</p><br />
<br /><br />
<p class="title"><i>Other</i></p><br />
<p><br />
We also found the <a href="http://biofuelpolicywatch.wordpress.com">BioFuel Policy Watch</a> blog and <a href="http://dglassassociates.wordpress.com">its associated blog</a> to be useful for general information. David Glass’s blog post on <a href="http://dglassassociates.wordpress.com/2013/09/22/regulation-of-industrial-use-of-algae-or-cyanobacteria-in-europe-part-1/">EU regulations for algae and cyanobacteria</a> is also a great starting point.<br />
</p><br />
<br />
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<tr><br />
<td colspan="3"><h3 class="title" id="acknowledgements">Acknowledgements</h3><br />
<p><br />
When we first asked the question, “can we put our bacteria on a roof?”, we didn’t envision giving such a detailed response. The proposition only reached its current form through repeated rounds of meetings with the students and supervisors, time spent reading EU and UK regulations and, most importantly, meetings with experts in biosafety at Reading. Gretta Roberts and Professor Jim Dunwell were very kind in giving up their time to answer all our questions, and we are very grateful for their input.<br />
</p><br />
<br /><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/File:Oscar_doing_stuff.jpgFile:Oscar doing stuff.jpg2014-10-18T00:34:51Z<p>Seafloor: </p>
<hr />
<div></div>Seafloorhttp://2014.igem.org/Team:Reading/PartsTeam:Reading/Parts2014-10-18T00:31:38Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br><br />
<html><br />
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#groupparts {text-align: center; margin-left: auto; margin-right: auto;}<br />
</style><br />
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<table width="70%" align="center"><br />
<br />
<!--Our parts--><br />
<h3 class="title"> Our parts</h3><br />
<br />
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<tr><td bgColor="#e7e7e7" colspan="3" height="1px"> </tr><br />
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<br />
<!--Parts Submitted to the Registry --><br />
Genetic modification in<br />
Synechocystis<br />
is done by modifying the chromosome,<br />
rather than inserting plasmids. Synechocystis is naturally transformable, and will<br />
undergo homologous recombination between its chromosome and a plasmid<br />
containing a homologous region. This means that plasmids for insertion or<br />
deletion need to have regions of ~500 to ~1000bp either side of the inserted<br />
sequence, making modifications time consuming or costly depending on your<br />
method of plasmid construction. We will therefore submit all our BioBricks for<br />
insertions and deletions to the registry. All of these will contain Kanamycin<br />
resistance for simple selection of transformants. <br />
<br /><br />
<br /><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6c/Matt_bg11plate_1.jpg" width=600px></center><br />
<br /><br />
<center>Above: Wild-type <i>Synechocystis</i> transformed with a plasmid carrying kanamycin resistance.</center><br />
<br /><br />
<br /><br />
The mechanism for each of the BioBricks is the same. They will undergo<br />
recombination with the region that they share homology with. Knockouts will<br />
undergo recombination with the specified gene, replacing it with kanamycin resistance. Insertions will undergo recombination with a region of the<br />
chromosome that is not important for the metabolic<br />
conditions we are using, and will insert the gene of interest along with a kanamycin resistance gene. <br />
<br /><br />
<br /><br />
We are creating 4 BioBricks that will be submitted<br />
to registry, consisting of 2<br />
deletions (<a href="http://parts.igem.org/Part:BBa_K1476003" title="Go to PsaD wiki page">PsaD</a> and <a href="http://parts.igem.org/Part:BBa_K1476000" title="Go to PilT1 wiki page">PilT1</a>) and 2 insertions (<a href="http://parts.igem.org/Part:BBa_K1476004" title="Go to PetF wiki page">PetF</a> and <a href="http://parts.igem.org/Part:BBa_K1476001" title="Go to PilA1 wiki Page">PilA1</a>). Background information on the aim of these parts can be<br />
found in our <a href="https://2014.igem.org/Team:Reading/Project" title="Go to project section">project section</a>. <br /> <br />
<br /> <br />
<br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<br />
<tr><td><h3 class="title"> Reading iGEM 2014 parts</h3></td><br />
<td></td><br />
<br />
</table><br />
</html><br />
<br />
<groupparts>iGEM013 Reading</groupparts><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/PartsTeam:Reading/Parts2014-10-18T00:31:15Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br><br />
<html><br />
<style type="text/css"><br />
#groupparts {text-align: center; margin-left: auto; margin-right: auto;}<br />
</style><br />
<!--main content --><br />
<table width="70%" align="center"><br />
<br />
<!--Our parts--><br />
<h3 class="title"> Our parts</h3><br />
<br />
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<tr><td bgColor="#e7e7e7" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!--Parts Submitted to the Registry --><br />
Genetic modification in<br />
Synechocystis<br />
is done by modifying the chromosome,<br />
rather than inserting plasmids. Synechocystis is naturally transformable, and will<br />
undergo homologous recombination between its chromosome and a plasmid<br />
containing a homologous region. This means that plasmids for insertion or<br />
deletion need to have regions of ~500 to ~1000bp either side of the inserted<br />
sequence, making modifications time consuming or costly depending on your<br />
method of plasmid construction. We will therefore submit all our BioBricks for<br />
insertions and deletions to the registry. All of these will contain Kanamycin<br />
resistance for simple selection of transformants. <br />
<br /><br />
<br /><br />
<center><img src="https://static.igem.org/mediawiki/2014/6/6c/Matt_bg11plate_1.jpg" width=900px></center><br />
<br /><br />
<center>Above: Wild-type <i>Synechocystis</i> transformed with a plasmid carrying kanamycin resistance.</center><br />
<br /><br />
<br /><br />
The mechanism for each of the BioBricks is the same. They will undergo<br />
recombination with the region that they share homology with. Knockouts will<br />
undergo recombination with the specified gene, replacing it with kanamycin resistance. Insertions will undergo recombination with a region of the<br />
chromosome that is not important for the metabolic<br />
conditions we are using, and will insert the gene of interest along with a kanamycin resistance gene. <br />
<br /><br />
<br /><br />
We are creating 4 BioBricks that will be submitted<br />
to registry, consisting of 2<br />
deletions (<a href="http://parts.igem.org/Part:BBa_K1476003" title="Go to PsaD wiki page">PsaD</a> and <a href="http://parts.igem.org/Part:BBa_K1476000" title="Go to PilT1 wiki page">PilT1</a>) and 2 insertions (<a href="http://parts.igem.org/Part:BBa_K1476004" title="Go to PetF wiki page">PetF</a> and <a href="http://parts.igem.org/Part:BBa_K1476001" title="Go to PilA1 wiki Page">PilA1</a>). Background information on the aim of these parts can be<br />
found in our <a href="https://2014.igem.org/Team:Reading/Project" title="Go to project section">project section</a>. <br /> <br />
<br /> <br />
<br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<br />
<tr><td><h3 class="title"> Reading iGEM 2014 parts</h3></td><br />
<td></td><br />
<br />
</table><br />
</html><br />
<br />
<groupparts>iGEM013 Reading</groupparts><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/TeamTeam:Reading/Team2014-10-18T00:30:18Z<p>Seafloor: </p>
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<h3 class="title" align="center">About Us</h3><br />
<p align="center">We are the University of Reading's first iGEM team. We are an entirely undergraduate team made up of students from both biology and engineering. As such we wanted a project which allow both disciplines to be utilised.</p><br />
</td><br />
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<h3 class="title" align="center">Meet the Team</h3><br />
</td><br />
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<br />
<table><br />
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<td width="45%"; valign="top" align="center"><br />
<h3>Matt Smith</h3><br />
<img src="https://static.igem.org/mediawiki/2014/2/2b/Matt.Smith.jpg" height="130px"></img><br />
<p>The resident biochemist. Lives off coffee and bagels. Enjoys free wifi. Talks in short sentences.</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Samuel Hardy</h3><br />
<img src="https://static.igem.org/mediawiki/2014/c/c4/Sam.Hardy.jpg" height="130px"></img><br />
<p>With a strong background in bacteriology and virology, Sam has a resilient interest in utilising some of our planet’s simplest life forms for good. Also the team photographer, activist and Doge enthusiast. Much wow.</p><br />
</td><br />
</tr><br />
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<td width="45%"; valign="top" align="center"><br />
<h3>Oscar Sanderson</h3><br />
<img src="https://static.igem.org/mediawiki/2014/8/85/Oscar.Sanderson.jpg" height="130px"></img><br />
<p>A key part of the lab team and the only colourblind member of the group. In charge of design and colour coordination. Naturally.</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Hannah Collard</h3><br />
<img src="https://static.igem.org/mediawiki/2014/e/eb/Hannah.Collard.jpg" height="130px"></img><br />
<p>Hannah is the Biomedical Scientist of the team. She does all the work.</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Sam Podmore</h3><br />
<img src="https://static.igem.org/mediawiki/2014/7/7a/Sam.Podmore.jpg" height="130px"></img><br />
<p>In charge of the engineering side of the project. Sam spends most of his time dreaming of Fourier transforms and consuming as much Iron Bru as humanly possible. He's also been caught drinking soup straight from the can (no, seriously).</p> <br />
<p><b>Most common saying: </b>"Yeah, I could model that"</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Andrew Moynihan</h3><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Andrew.Moynihan.jpg" height="130px"></img><br />
<p>With a background in chemistry and programming, Andrew joined the team as an engineer, <br />
although he is able to support the team in the lab as well. Tasked with most of the design <br />
and implementation of the web services, he remains the first point of contact for <br />
information on the wiki.</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Domonic Falla</h3><br />
<img src="https://static.igem.org/mediawiki/2014/6/6e/Dom_falla_photo.png" height="130px"></img><br />
<p>The youngest member of the team and yet also generally regarded as the most attractive <br />
member. His youth belies his precocious wisdom, and correspondingly he is looked to as <br />
very much the spiritual leader of the team.</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Jonathan Pennell</h3><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/IMG_7283.png" height="130px"></img><br />
<p>Is a microbiologist that isn’t a big fan of lab work, nor people really. After recently <br />
discovering the field that is Bioinformatics he generally tends to stick around computers <br />
more so than living things, he understands them. Trusts them... Talks to them...</p><br />
</td><br />
</tr><br />
<tr><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Eleanor James</h3><br />
<img src="https://static.igem.org/mediawiki/2014/9/9f/Eleanor.png" height="130px"></img><br />
<p>The requisite Welsh person, generally regarded as the life and soul of the synthetic biology <br />
party. Loves riding horses and rowing, but hates slow Internet. Don’t mention sheep.</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Calum Brown</h3><br />
<img src="https://static.igem.org/mediawiki/2014/e/e7/Jonathan.Pennell.jpg" height="130px"></img><br />
<p>Calum is the cynical member of the team. With a background in physics, robotics and <br />
electronics, Calum doesn’t understand humour, and/or punctuation. As an engineer he has <br />
the social skills to interact with a wide range of people and as such he forms part of the <br />
public outreach side of the project.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</br></br></br></br><br />
<br />
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<td > <br />
<h3 class="title" align="center">Advisors</h3><br />
</td><br />
</tr><br />
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<table><br />
<tr><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Lauren Vallance</h3><br />
<img src="https://static.igem.org/mediawiki/2014/a/a6/IMG_7559.png" height="130px"></img><br />
<p>"Starbucks?".</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Joel Potts</h3><br />
<img src="https://static.igem.org/mediawiki/2014/b/b8/Joel_Potts_Photo.png" height="130px"></img><br />
<p>Advisor to the engineers. Helps with all things programming...if it's in Ruby.</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</br></br></br></br><br />
<br />
<tr><br />
<td><br />
<h3 class="title" align="center">Instructors</h3><br />
</td><br />
</tr><br />
<br />
<table><br />
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<td width="45%"; valign="top" align="center"><br />
<h3>Jaroslaw Bryk</h3><br />
<img src="https://static.igem.org/mediawiki/2014/0/09/Jarek_circle_small.png" height="130px"></img><br />
<p>Committed to synthetic biology education. Currently fighting a bagel addiction. Gibson Assembly FTW.</p><br />
</td><br />
<td width="45%"; valign="top" align="center"><br />
<h3>Slawomir Nasuto</h3><br />
<img src="https://static.igem.org/mediawiki/2014/1/18/Sse-staff-slawomir-nasuto.png" height="130px"></img><br />
<p>Professor at The University of Reading within the School of Systems Engineering. Interested in neuroscience, neuroanatomy and brain computer interfaces</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</body><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Template:HeadTemplate:Head2014-10-18T00:28:54Z<p>Seafloor: </p>
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<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Human_Practices';">Human Practices</td><br />
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<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Lab Work';">Lab book</td><br />
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<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Protocols';">Protocols</td><br />
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<td align ="center" bgColor=#A2D185> <a href="https://2014.igem.org/Team:Reading"> <img align=centre src="https://static.igem.org/mediawiki/2014/5/56/IGEM-Logo-Black_small.png" width="55px"> </a></td> </tr><br />
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</html></div>Seafloorhttp://2014.igem.org/Template:HeadTemplate:Head2014-10-18T00:27:16Z<p>Seafloor: </p>
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<br />
<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Lab Work';">Lab book</td><br />
<br />
<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Protocols';">Protocols</td><br />
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<br />
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</html></div>Seafloorhttp://2014.igem.org/Template:HeadTemplate:Head2014-10-18T00:27:05Z<p>Seafloor: </p>
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<br />
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<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Parts';">Parts</td><br />
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<br />
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<br />
<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Protocols';">Protocols</td><br />
<br />
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<br />
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</html></div>Seafloorhttp://2014.igem.org/Template:HeadTemplate:Head2014-10-18T00:26:26Z<p>Seafloor: </p>
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<br />
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<br />
<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Lab Work';">Lab book</td><br />
<br />
<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Protocols';">Protocols</td><br />
<br />
<td align="center" height ="20px" width="90px" onMouseOver="this.bgColor='#9AC77F'" onMouseOut="this.bgColor='#A2D185'" bgColor=#A2D185 onClick="document.location.href='https://2014.igem.org/Team:Reading/Attributions';">Attributions </td><br />
<br />
<td align ="center" bgColor=#A2D185> <a href="https://2014.igem.org/Team:Reading"> <img align=centre src="https://static.igem.org/mediawiki/2014/5/56/IGEM-Logo-Black_small.png" width="55px"> </a></td> </tr><br />
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</html></div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:24:00Z<p>Seafloor: </p>
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<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
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<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
</ol><br />
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<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generate energy.</br></br><br />
In our cells, microorganisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our cyanobacterial cell uses solar energy to stay alive, only needing an inorganic carbon source (such as carbon dioxide), water, and low levels of minerals to continue to be productive.<br />
</br></p><br />
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<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
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<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
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<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
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<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiment, <i>Synechocystis</i> grew as expected; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<br /><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
<br /><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:22:33Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generate energy.</br></br><br />
In our cells, microorganisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our cyanobacterial cell uses solar energy to stay alive, only needing an inorganic carbon source (such as carbon dioxide), water, and low levels of minerals to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<br /><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
<br /><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:20:36Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, microorganisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our cyanobacterial cell uses solar energy to stay alive, only needing an inorganic carbon source (such as carbon dioxide), water, and low levels of minerals to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<br /><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
<br /><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:15:55Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Microorganisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our cyanobacterial cell uses solar energy to stay alive, only needing an inorganic carbon source (such as carbon dioxide), water, and low levels of minerals to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<br /><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:10:37Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<br /><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:10:03Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<br /><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-18T00:09:41Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long-term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<tr><br />
<td><br />
<p><br />
We wanted to plan out the system we would set up on each roof. To do this, we envisioned how it would work on a rooftop in our university or on the roof of a person's house. The hypothetical situation we considered is panels large enough to fit on an average roof. The system would be set up by using large, flat, pre-sterilised containers, contained the sterile carbon fibre electrodes. Cyanobacteria would be cultured in a separate facility, and added to the anodic chamber of the container once a suitable density was reached. The electron acceptor, which would probably be potassium ferricyanide, would be added to the cathodic chamber. The two chamber sit horizontally in the panel, with the anode on top. This allows light to penetrate the panel and reach our cyanobacteria, which then donate electrons to the carbon fibre anode that they are growing on. A proton-permeable membrane separates the two compartments, as with a normal fuel cell.<br />
</p><br />
<p><br />
The system would be sealed and transported to the site (either the university or a private property), so that our system is completely sealed when it leaves our factory. As nutrients in the media, and CO<sub>2</sub>, would decrease over time, we may need to use a drip in/drip out system, where users would attach a fresh tank of minimal media to drip in, and a waste tank fills up. Waste disposal is a key issue when considering risks associated with GM work. Our system would use a filter, so that the waste water was completely GMM-free and could be disposed of down a normal drain. The filter would need to be replaced at certain points (with the old one being collected for sterilisation) to prevent it clogging. Furthermore, long-term installations would need a source of low-level CO<sub>2</sub>. Air level is sufficient for this. We would need to pump sterile air through the panel to maintain low CO<sub>2</sub> conditions; alternatively, sodium hydrogen carbonate could be added in low amounts to the medium prior to shipping. The panel may also need an access point where a maintenance worker could easily, but securely, take a sample from the panel to check that biological controls, such as engineered auxotrophy, were still functional.<br />
</p><br />
</td><br />
</tr><br />
<br />
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<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:43:02Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
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<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<i>Synechocystis</i> can be frozen for transport or just kept alive on plates for shipping. The benefit of using microorganisms is that large liquid batches do not need to be transported; due to their reasonably fast growth (doubling time is around 12 hours), cultures could grow up in a matter of weeks. This allows scalability based on the needs and available space at the target location. Even in non-remote areas, our fuel cell can out-perform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:35:45Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="950px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
</p><br />
<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:35:29Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="945px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
</p><br />
<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:34:53Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="900px"></td><br />
</tr><br />
</table><br />
<table><br />
<tr><br />
<td><br />
<p><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
</p><br />
<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:34:01Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="900px"></td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
</p><br />
<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:33:03Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
<br />
</td><br />
<br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- Fuel Cell Basics --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
<br />
<!-- Subsection <br />
<br /><br />
<br /><br />
<p class="title" id="exampleone"><i>Sample Subsection Number One</i></p><br />
<p>This is how an example subsection could be formatted.</p><br />
</p><br />
</td><br />
</tr><br />
--><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
</table><br />
<br />
<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr><br />
<td colspan="3" height="15px"><br />
<br><br />
<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.<br />
</p><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/20/Pond_sample_cyano.jpg" width="900px"></td><br />
<p><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
</p><br />
<br />
</p><br />
</td><br />
</tr><br />
<br />
<!-- Spacer line--><br />
<tr> <td colspan="3" height="15px"> </td></tr><br />
<tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr><br />
<tr> <td colspan="3" height="5px"> </td></tr><br />
<br />
<!-- References Section --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
<br />
</table><br />
</td><br />
</tr><br />
<br />
</html><br />
<br />
{{Tail}}</div>Seafloorhttp://2014.igem.org/File:Pond_sample_cyano.jpgFile:Pond sample cyano.jpg2014-10-17T23:32:14Z<p>Seafloor: </p>
<hr />
<div></div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:27:08Z<p>Seafloor: </p>
<hr />
<div>{{Head}}<br />
<br><br><br><br><br><br><br><br><br><br />
<html><br />
<br />
<table><br />
<br />
<br />
<tr><br />
<td><br />
<br />
<!-- Fuel Cells --><br />
<tr><td><h3 class="title"> Introduction</h3></td><br />
<td> <h3 class="title" id="intro">Contents</h3></td><br />
</tr><br />
<br />
<!-- Introduction --><br />
<tr><br />
<td width="80%" valign="top"> <br />
<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
<br />
<br><br />
</td><br />
<td width="36%" valign="top"><br />
<br />
<!-- Contents - add stuff here to add to the contents page --><br />
<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
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<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
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<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
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<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
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<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using <a href="http://www.sodis.ch/index_EN">the SODIS method</a>, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.</br></br><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
<br />
</p><br />
http://www.sodis.ch/index_EN<br />
</p><br />
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<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Fuel_CellTeam:Reading/Fuel Cell2014-10-17T23:24:45Z<p>Seafloor: </p>
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<td> <h3 class="title" id="intro">Contents</h3></td><br />
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<p><br />
All fuel cells adhere to a basic design. On this page we'll introduce <a href="#system">how they work</a>, then show you <a href="#ours">some of our own</a>. You can also <a href="#panel">check out plans for making a large rooftop panel</a>, and head over to the human practices page to <a href="https://2014.igem.org/Team:Reading/Human_Practices">see how we'd pass the regulatory requirements</a>.</p><br />
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</td><br />
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<ol><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#system">Fuel Cell Design</a></li><br />
<ul><br />
<li><a href="#exampleone">Example</a></li><br />
</ul><br />
<li><a href="#ours">Our Fuel Cells</a></li><br />
<li><a href="#panel">Scaling it up</a></li><br />
<li><a href="#references">References</a></li><br />
</ol><br />
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<td colspan="3"><h3 class="title" id="system">Fuel Cell Design</h3><br />
<p bgColor=B2E592><br />
<p><br />
A fuel cell is different from other cells (like batteries) in that there is a continuously replenished source of energy involved; the most common example of a fuel cell is a Hydrogen Cell, which takes continuous inputs of pure Hydrogen and atmospheric Oxygen to create water, generating energy.</br></br><br />
In our cells, Micro-organisms are constantly doing work to generate energy, but need fuelling over time. Yeast cells are more traditional, requiring sugars to keep alive and generating energy. On the other hand, our Bacterial cell uses solar energy to stay alive, only needing a carbon source (such as carbon dioxide) to continue to be productive.<br />
</br></p><br />
<br />
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<!-- Info and pictures of our fuel cell --><br />
<table><br />
<tr><br />
<td colspan="3"><h3 class="title" id="ours">Our Fuel Cells</h3><br />
<p><br />
A look at some of our fuel cells, whether they're cyanobacterial, yeast, or just plain ol' mud.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/2/2a/Fuel_cell_small.jpg" width="450px"></td><br />
<td><img id="methods" align=right src="https://static.igem.org/mediawiki/2014/6/66/Fuel_cell_large_2.jpg" width="450px"></td><br />
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<video width="100%" controls src="https://static.igem.org/mediawiki/2014/8/88/Cyanocell_with_sound.mov" /><br />
</td><br />
</tr><br />
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<br />
<!-- The Rooftop Panel --><br />
<tr><br />
<td colspan="3"><h3 class="title" id="panel">Scaling it up</h3><br />
<p> <br />
At a laboratory scale, our cell gets rapidly outperformed by an AA battery. However, our intent was to create a fuel cell that can outperform standard solar cells on cost, ease of use and maintenance, with the aim of providing easy power sources for incredibly remote locations. Since our cells work with minimal media, it is possible to use sterilised pond water as the base, meaning you don't have to transport perfect media to the desired location. We have tested this by UV-sterilising pond water using the x method, then passing the pond water through a rudimentary filter. UV-sterilisation is done using normal PET plastic bottles that would be easily available in less-developed countries. Rags or old pieces of clothing could be used as a filter to remove larger pieces of debris or plant material. We used PET bottles and paper towels, then added our cyanobacteria to the water. We would expect pond water to contain the low levels of iron and minerals required by our organism, though not necessarily in optimal concentrations, allowing <i>Synechocystis</i> to grow. In our experiement, our cyanobacteria did grow; you can our pond water cultures thriving below.</br></br><br />
Synechococcus Bacteria can be dried and packaged for transport, meaning no live cultures need to be maintained, and due to the rapid growth of micro-organisms a small sachet will be able to fill any size container in a few days. This allows scalability based on the needs and available space at the target location.</br></br><br />
Even in non-remote areas, our fuel cell can outperform standard solar cells on price, paying for their initial investment in just 30% of the time. With no high cost components, long term maintenance is also cheap and easy.<br />
<br />
</p><br />
<br />
</p><br />
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<td colspan="3"><h3 class="title" id="references">References</h3><br />
<p>Here are the references.</p><br />
</td><br />
</tr><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Human_PracticesTeam:Reading/Human Practices2014-10-17T22:48:59Z<p>Seafloor: </p>
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<h3 class="title" id="summary"> Regulatory challenges of rooftop installations</h3><br />
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<p><br />
An important part of iGEM is thinking about the wider impact of your project. We considered whether it would be possible to set up our cyanobacterial solar panels on roofs at Reading or on people’s houses. This meant coming up with a <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">design for a larger photovoltaic cell</a>, considering the biosafety issues involved, and what regulatory challenges we would face.<br />
</p><br />
<p><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/3/30/Cyano_cultures.jpg" width="800px"></p><br />
</td><br />
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<p><h3 class="title"> Contents</h3></p><br />
<p><br />
<ol><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#levels">Levels of Regulation</a></li><br />
<li><a href="#eu">EU Regulations</a></li><br />
<ul><br />
<li><a href="#euintro">Introduction</a></li><br />
<li><a href="#eucontained">Contained Use</a></li><br />
<li><a href="#eudelib">Deliberate Release</a></li><br />
</ul><br />
<li><a href="#uk">UK Regulations</a></li><br />
<ul><br />
<li><a href="#ukintro">Introduction</a></li><br />
<li><a href="#ukcontained">Contained Use</a></li><br />
</ul><br />
<li><a href="#other">Other Regulations</a></li><br />
<li><a href="#safety">Biosafety</a></li><br />
<li><a href="#conc">Findings and Conclusions</a></li><br />
<li><a href="#road">The Roadmap</a></li><br />
<li><a href="#resources">Resources</a></li><br />
<li><a href="#acknowledgements">Acknowledgements</a></li><br />
</ol><br />
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<p><br />
Creating a cyanobacterial photovoltaic cell and getting it installed on a roof at a university presents a number of challenges. The design of the system is the first aspect to consider. We look into design and cost of the parts on the Fuel Cell page. Then there are European Union (EU) and government regulations and, in the case of Reading, several boards and internal committees through which applications would have to pass. We consider these in this section. Each of these raises questions about biosafety, such as potential effects of the escape of our organism into surrounding environments. In addition to using our technology at our own university, we also considered commercialising the technology and installing it on people’s houses. This opens up a new realm of issues, such as getting our energy source classed as renewable according to the EU’s Renewable Energy Directive (RED), and getting permission for having GMOs on many distinct properties. These wider problems are also reviewed here.<br />
</p><br />
<p><br />
Many sections of our report will be applicable to other teams considering contained use of GMOs, and we hope future teams will benefit from our research. The EU section will be particularly relevant to other EU member states, as the EU regulations form the common minimum requirements for each country. In general, this page should guide teams considering biosafety issues associated with cyanobacteria; there are currently no reviews of biosafety in synthetic biology of cyanobacteria that we are aware of. We finish with a roadmap for those thinking about whether they could commercialise a genetically modified microorganism (GMM)-containing system, especially as a renewable fuel source.<br />
</p><br />
<p><br />
It should be noted that the report mainly refers to contained use of GMMs. Though our system is contained, parts of it could be considered to overlap with deliberate release. We have therefore focussed on regulations pertaining to contained use, but have referred to those on deliberate release where our system could potentially fall under its purview. Due to this relevance, and partly time due to time constraints, we have not exhaustively considered deliberate release or contained use categorised as class 2 or above.<br />
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<td colspan="3"><h3 class="title" id="levels">The Different Levels of Regulations</h3><br />
<p><br />
At the highest level, the Cartagena Protocol on Biosafety covers living modified organisms (LMOs) and their transport across borders. This is an international United Nations agreement that has been in place since 2003, and is implemented in the EU by <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>. Below that, the EU issues “directives” on genetically modified organisms (GMOs) that must be implemented by all EU member states. For contained use, only the state’s regulations need to be considered; there is no involvement at the EU level. For deliberate release, rules are much more complicated, involving notification of the European Commision (EC), and will not be covered here. In the UK, the EU directives are implemented by the Department for Environment, Food and Rural Affairs (DEFRA) and the Health and Safety Executive (HSE). Finally, at Reading we would have to pass at least 3 committees - including the sub-committee for biological safety, the project committee, and the environmental committee - in addition to getting approval from the building manager.<br />
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<td colspan="3"><h3 class="title" id="eu">EU Regulations</h3><br />
<p class="title" id="euintro"><i>Introduction to EU Regulations</i></p><br />
<p><br />
The two EU directives concerning our plans are the directive 2009/41/EC on contained use and the 2001/18/EC directive on deliberate release of GMOs. Of these, the contained use directive is probably most appropriate. The 2009/41/EC directive defines contained use as: “any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment”, in Article 2(c).<br />
</p><br />
<p><br />
By contrast, the 2001/18/EC directive defines deliberate release as: “any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment”, in Article 2(3).<br />
</p><br />
<p><br />
By comparing these, and reviewing <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the proposed implementation of our idea</a>, we can see that we would most likely fall under the contained use directive because of our suggested containment measures. Our technology will use all of the activities specified by contained use, and implement appropriate safety measures. Based on this, we shall chiefly address contained use regulations, but mention rules on environmental release that are significant.<br />
</p><br />
<br /><br />
<p class="title" id="eucontained"><i>EU: Contained Use</i></p><br />
<p><br />
In summary, class 1 contained use requires: <br /><br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment</li><br />
<li>classification of the risk of the GMM according to the assessment</li><br />
<li>appropriate containment</li><br />
<li>notification of the relevant authority</li><br />
<li>an emergency plan for accidental release</li><br />
</ul><br />
</p><br />
<p><br />
We shall explore each of these points in further detail.<br />
</p><br />
<p><br />
Article 4 defines one of the main requirements for contained use: for a risk assessment to be carried out (Article 4(2)), in accordance with the guidelines in Annex III, with the aim of classifying the GMM. The criteria include assessing the potential to cause disease, effect on the environment (Annex III (A1)), and harmful effects of the genetic material, recipient, donor, vector and final GMM (Annex III (A2)). The severity of these issues and the chance of them happening must also be analysed. As a non-pathogenic organism, capability of causing disease is not relevant to our organism. The most germaine section is in Annex III (A1), which lists considering “deleterious effects due to establishment or dissemination in the environment” and “deleterious effects due to the natural transfer of inserted genetic material to other organisms.” Annex III (B7) goes on to require that the proposed use of the microorganism be combined with the above assessment in assigning it to a class. “Non-standard operations” is mentioned as affecting classification (Annex III (B7 iii)); this term is ambiguous, but may encompass our suggestion of having GMMs on roofs of private properties. <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">Our drip-in/drip-out system</a>, with the filter needing autoclaving upon replacement, may also fall under non-standard use. This would need to be taken into consideration if attempting to use our technology commercially. From this, it is clear that our GMM belongs in class 1 (as defined in Article 4(3)), so requires level 1 containment measures, though any doubt raised from our “non-standard operations” might cause a more strict classification (Article 4(4)). For those wishing to classify their organism, Directive 2000/54/EC can be referred to, or classification systems of the specific country.<br />
</p><br />
<p><br />
The risk assessment has a particular focus on waste disposal (Article 4(5)), making our drip-out waste disposal system very important. As removing the filter from the panel might be a source of accidental release, careful planning of waste management should be high on our priority list. The final risk assessment must be given to the competent authority (Article 4(6)) - HSE in the case of the UK. Containment measures are defined in Annex IV.<br />
</p><br />
<p><br />
Article 6 poses a potential issue. It requires notifying authorities upon contained use at each new property. While this is reasonable for single use at the university, our idea of having installations on separate houses would mean giving the information listed in Annex V for each site, including the risk assessment, which individuals are responsible for supervising, and a description of the premises. This would mean that the risk assessment must be sufficiently comprehensive to envision all potential environments where an installation may be set up, and would mean extra administration work for our organisation. It may be that, in the future, EU directives would need to be altered in order to make GMM technologies like ours more easily available for public benefit. Deliberate release regulations already contain a separate section for commercial use, and contained use may be separated this way in the future too.<br />
</p><br />
<p><br />
For class 1 organisms, no further notification is needed before commencing with contained use (Article 7). For higher risk classes more information is needed; as our organism is only class 1 we will not consider this, but rules can be found in Articles 8 and 9.<br />
</p><br />
<p><br />
Further thought should be given to the minimum containment measures stipulated in Annex IV, and whether our system meets these conditions. There are different requirements given for different potential situations. Our proposition would most likely fall under “Containment and other protective measures for other activities” for the panel itself, but other containment procedures would need to be reviewed for labs where genetic modification is done and areas where GMMs are cultured. Almost all containment options for class 1 organisms in this category are optional according the Annex IV. It is likely that the level of containment assumed for this category is more severe than in our proposition (i.e. the EC has assumed all contained use will occur inside a building). As such, we should expect to see some or all of the containment measures to be required, rather than optional, for our project. <br />
</p><br />
<p><br />
Beyond the obvious physical containment, there are several possible containment measures we could be required to enforce. This includes control of aerosols during “addition of material to a closed system or transfer of material to another system”. This would include transferring cyanobacteria to the fuel cell, which would be done in a separate contained facility, and during the removal of waste or the waste filter <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">(see design section)</a>, which would have to be done on site. The latter is one of the biggest issues our project could face.<br />
</p><br />
<p><br />
Inactivation of waste containing GMMs is also listed as optional, but could potentially be required. Furthermore, the air leaving the system, assuming filter-sterilised air is bubbled through our panel, could have to be filtered to prevent or minimise release. The only point already required for class 1 work is that personnel wear protective clothing.<br />
</p><br />
<p><br />
According to Article 13, an emergency plan is required. This must be made available to the public, relevant bodies and authorities, and other concerned EU member states. The plan is required in case containment measures fail, leading to “serious danger, whether immediate or delayed, to humans outside the premises and/or to the environment”. No information is given on how extensive this plan should be, and no minimum requirements are given. Member state legislation must therefore be consulted for any rules on how the plan must be structured.<br />
</p><br />
<p><br />
Finally, it should be noted that member states may consult the public on the proposition if they decide it is relevant (Article 12).<br />
</p><br />
<br /><br />
<p class="title" id="eudelib"><i>EU: Deliberate Release</i></p><br />
<p><br />
Below is outlined some of the salient points from the EU directive on deliberate release. These may be useful to other teams reviewing regulations. The key points are:<br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment is required (Article 4; Annex III)</li><br />
<li>regulations are different for commercial and non-commercial use</li><br />
<li>for non-commercial GMO work (Part B):</li><br />
<ul><br />
<li>parties must give a risk assessment and monitor use and effects</li><br />
<li>the authority can tell the public</li><br />
<li>approved uses must be reported to the EU</li><br />
</ul><br />
<li>for commercial GMO work (Part C):</li><br />
<ul><br />
<li>parties must notify the relevant authority before placing the product on the market</li><br />
<li>putting it on the market is defined as making it available to 3rd parties</li><br />
<li>the authority produces an “assessment report”</li><br />
<li>this is given to the applicant, the EC and EU member states</li><br />
<li>decisions apply throughout the EU</li><br />
<li>the public must be notified</li><br />
<li>as of June 2014, <a href="http://ec.europa.eu/food/plant/gmo/legislation/future_rules_en.htm">member states can restrict or ban GMOs in their country</a> that have been approved for all states</li><br />
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<td colspan="3"><h3 class="title" id="uk">UK Regulations</h3><br />
<p class="title" id="ukintro"><i>Introduction to UK Regulations</i></p><br />
<p><br />
<p><br />
The EU regulations are useful to us as they provide a baseline level of regulation we can expect if we try to implement our technology anywhere in the EU. In each EU member state, it is ultimately that country’s regulations which we must abide by. We will now consider what the regulations are like the UK. <br />
</p><br />
<p><br />
In general, they are slightly stricter than the basic EU regulations. The main regulations are <a href="http://www.hse.gov.uk/pubns/priced/l29.pdf">the HSE Contained Use regulations</a>, which are newly updated for 2014, and the accompanying SACGM Compendium of Guidance, which has yet to be updated to meet the new Contained Use document. Along with this, there are <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the regulations on deliberate release</a> from 1997, <a href="http://www.legislation.gov.uk/ukpga/1990/43/contents">section 108(1) of the Environment Protection Act </a> from 1990, and <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the Genetically Modified Organisms Regulations </a> (1996) that are related to GMOs. The latter three are all focussed on environmental release, so won’t be covered here. Regulations are upheld by the HSE and DEFRA. <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">The HSE website</a> can be consulted for all other regulations that might relate to the use of GMOs.<br />
</p><br />
<br /><br />
<p class="title" id="ukcontained"><i>UK Regulations: Contained Use</i></p><br />
<p><br />
The essential requirements for contained use in the UK are in the line with EU rules; we need to carry out a risk assessment and classify our organism, and notify the HSE before commencing GM work. So far we have only talked about the sites where the panels will be installed, but we will also have to consider regulations for handling, transport, work area decontamination, inactivation of GMMs and their disposal (including waste management). While the GMMs’ safety would need to be assessed by HSE, the system containing them, our panel, will need to be tested for leakages, with this evidence submitted to DEFRA.<br />
</p><br />
<p><br />
The definition given for contained use in Part 1, regulation 2 is “an activity in which organisms are genetically modified or in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers, or any combination of such barriers, are used to limit their contact with, and to provide a high level of protection for, humans and the environment”. Also in part 1, regulations 26 specifically tells us that commercial disposal of waste containing GMOs also falls under contained use. The contained use definition is similar to the EU definition, but is slightly more specific about what the containment measures must entail. In Part 1, paragraphs 24 and 45 give examples of what the barriers for our system might be expected to be. Physical could include a container, which would be the panel itself in our case. Chemical barriers may cover inactivation before waste disposal, and biological would include attenuating characteristics that debilitate the organism so that it is “rendered unable to survive outside of a specialised environment”. These barriers are discussed further in the safety page.<br />
</p><br />
<p><br />
The first requirement to consider is the risk assessment. For GMMs this is covered in Part 2, regulation 5, with more details on the assessment in Schedule 3 (Part 2). More emphasis is placed on risk to human and health and environment than in EU regulations. Regulation 5, paragraph 43, also answers questions we raised in the “EU: Contained Use” section about whether we could apply the same risk assessment to multiple sites with the same roof installation: “Where the contained use is identical at the multiple sites (eg in a clinical trial), the same risk assessment may apply to all the sites”. However it does point out that local changes in practices need to be taken into account. For us, practices would remain the same, but the surrounding environment may be different (e.g. there may be a pond at one property, where our organisms could theoretically survive). What the risk assessment must encompass is introduced in paragraph 44. In short, we must outline our plans, potential harmful effects, the chance of them occurring and their severity, and how we’ll deal with waste. These topics are covered in the safety page, where we look at each of our mutations; waste disposal is covered in <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the Fuel Cell page</a>.<br />
</p><br />
<br /><br />
<p><img align=centre src="https://static.igem.org/mediawiki/2014/7/77/Containment_lab_1.jpg" width="800px"></p><br />
<br /><br />
<p><br />
It is clear that the detail needed in the risk assessment is partly defined by how well-understood the microorganism and mutations are. Although our organism is clearly a non-pathogenic, non-hazardous class 1 organism, it is not as well understood as Escherichia coli K-12, for example, and the genes are not ones that are commonly used. They do not have a strong history of safe use, like green fluorescent protein (GFP). All our mutations have been done before, however, while measuring for different endpoints, so literature is available for reference on the effects of our mutations. From paragraphs 52 and 53, we know that the classification of the work changes to reflect the level of containment needed. When considering EU regulations, we were unsure of the extent of containment required, as our organism is class 1, but is used in unusual and potentially problematic environments. Under UK regulations, it appears our work could be reclassified to class 2 if we deem the containment measures for class 2 work to be desirable. This brings in a previously unforeseen hurdle: our work may be relabelled as higher than class 1 because of the containment measures needed on private properties with no trained personnel. We will continue to assume our work is class 1, but mention class 2 rules where appropriate.<br />
</p><br />
<p><br />
In summary, for the risk assessment we must identify hazards, assign appropriate containment measures, then reclassify our work based on these (if necessary). These instructions are similar to, but more detailed than, those for EU member states in general.<br />
</p><br />
<p><br />
According to regulation 8, we need to obtain advice from a person or committee on the risk assessment. If our organism were reclassified as class 2, this would have to be a biological safety committee. At Reading this would not be an issue, as there is already a committee from which we could obtain advice. If the classification remained as class 1, our meeting with Gretta Roberts and Professor Jim Dunwell, who have advised us with regards to regulations and safety, should be sufficient.<br />
</p><br />
<p><br />
Regulation 9 requires notification of premises to be employed for contained use. We must first submit the information in Schedule 5, then wait 10 days for a response. A single notification may include more than 1 premises in situations where more than one premise is owned by the same company, so it’s possible that multiple sites could be asked about at once. This would reduce the cost of notifying HSE, which is currently <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£472 for class 1</a> and <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£943 for class 2</a>. However, if our organism still falls under class 1, we only need to submit a summary of the risk assessment, details on waste management and the advice we received during the risk assessment, and confirmation that relevant authorities will be notified of the emergency plan. The rest of the information needed is basic details like the address of the premises. For class 2 work, regulation 10 should be consulted.<br />
</p><br />
<p><br />
Part 3 outlines practices that must occur for contained use. Regulations 18, 19, 21 and 22 are all relevant to different stages of our plans. Sections relevant specifically to the panels include paragraphs 107-109, which tell us that containment measures must be tested; this may involve checking each panel for defects or leaking before deployment, and possibly visiting sites at intervals to check they are still functioning correctly (paragraph 108). This could mean simply looking over the panel for any cracks or leakages to check for physical containment. Checking that biological containment is still in place, for example by checking that cells cannot directly transfer or uptake DNA after the pilT1 mutation, may involve taking a sample from panels. This itself means removing liquid containing GMMs and transporting it back to the lab for testing transformation efficiency; we would have to ensure that taking any liquid samples would not have any risks of accidental release. It is unlikely, but possible, that we could be required to check for our GMM in the surrounding environment to ensure there had been no release (paragraph 111).<br />
</p><br />
<p><br />
Regulation 19 specifically covers containment measures for GMMs. Containment measures must be reviewed “at regular intervals” (paragraphs 128 and 129), which must occur more frequently for non-standard work. According to paragraph 134, our organism does not fit the criteria for class 1 work that does not require waste inactivation, because our organism does not contain “multiple disabling mutations”, so waste from our panel must be inactivated. According to 135, it is acceptable to take the filter to another location and autoclave it, assuming steps are taken to make sure storage and transport are safe, and that the process is effective at inactivating our GMM.<br />
</p><br />
<p><br />
We must follow Schedule 8, Part 2, Table 2 for containment measures. As with our assessment of EU containment requirements, we have chosen the “other” section, as this seems most appropriate. Here the only absolute requirement is that personnel must wear work clothing. Measures that might be required, if deemed so by the risk assessment, include: physical separation, control of aerosols (as discussed in the EU contained use section), inactivation of waste or removed fluid, and control for spillage. The last of these is the only one not fully considered so far in our design.<br />
</p><br />
<p><br />
Emergency plans are discussed in regulation 21. According to paragraph 139, however, “an emergency plan should only be prepared for work with organisms that pose the highest hazards to humans or the environment.” Although our organism is low hazard, the risk is perhaps higher because the GMMs are not contained in a facility. As only hazard is mentioned, it is possible that we would not have to draw up an emergency plan (for our organism the hazard is low, but the risk slightly higher as it is not contained in a building). Furthermore, only those on the premises would be exposed to our non-dangerous GMM, but paragraph 139 only requires an emergency plan when the health or safety of those outside the premises is in danger. This probably assumes the work is a designated building with trained personnel on site, though. Given this, and our non-standard plans, it would be prudent to anticipate an emergency plan being required. If this were the case, it must be submitted along with our contained used application to HSE (paragraph 140). Paragraph 141 covers the requirement for an emergency plan; those “on the site affected by the plan” should know the plan, so residents in houses where a panel is installed, or building managers for a university building, would have to be familiar with the plan (paragraph 142). The plan must also be publicly available (paragraph 143), as we know from the EU requirements. Although we have primarily talked about the risk to the environment of our GMMs, we should also consider the risks of releasing potassium ferricyanide to the environment (a part of our solar panel), like how much would be released, any risks to human health or safety, and any risks to the environment.<br />
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<p><br />
We’ve covered the main EU and UK regulations regarding the installation of our actual panels, but there are many other regulations relevant to our plans. Contained use regulations are the main ones, which would cover the genetic modification of our organisms in a lab, the transport of our organisms to a facility where they can be grown up, that facility itself, transport to houses or the university, and the property where the panel is installed. There are other regulations that could come into play, however. This includes rules on the <a href="http://www.hse.gov.uk/cdg/introduction.htm">carriage of dangerous goods</a>, which might include our GMMs or potassium ferricyanide. Furthermore, transport regulations for crossing borders in the EU, which might occur if we export cultivation of our GMMs to another country, would mean we must adhere to EU GMO border rules in <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>, which implements the Cartagena Protocol.<br />
</p><br />
<p><br />
At Reading, we would also have to submit an application to at least 3 committees before getting our proposal approved, and speak to the building manager for each building we would want to get our panel put on. Although the committees sound like more regulatory hurdles, the university safety officers would contact HSE for us, and we would supply all our information to the safety officers, making the process much easier. Furthermore, the biological safety committee would be the committee we would contact for the expert advice required when carrying out a risk assessment, and all the buildings at the university count as one private property, for which only one application to HSE needs to be made.<br />
</p><br />
<p><br />
Commercialisation of our product would mean selling it as a new source of renewable energy. Renewable fuel sources are subject to other rules in the EU, which stipulate criteria that must be met for a source to be labelled as “renewable”. The Fuel Quality Directive (FQD) is relevant to renewable fuels for transport, such as biofuels, and the Renewable Energy Directive (RED) is pertinent to other energy sources that wish to be labelled as renewable; ours could fall under the latter. The requirements in RED are essentially requirements for EU member states, but in order for the standards to be met, it is individual companies that ultimately must comply. The RED is enforced by the European Commission Directorate General for Energy. The requirements include showing a reduction in greenhouse gas emissions over the course of the fuel’s production, and using life cycle analysis (LCA) methods to calculate the “carbon intensity” of our energy source. Our “sustainability analysis” must encompass other features beyond an LCA. To meet the specifications in the RED, we must check 12 independent factors of our energy source (including its production and transportation), and have this verified by a third party. The analysis method must be approved by the EU.<br />
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<p><br />
We have now reviewed the safety requirements at the EU and UK levels. The exact steps for the UK risk assessment are outline in Section 3, part 2, of the Contained Use regulations. To meet the requirements for the UK, we must provide information on our organism. First, we will have to prove our organisms are less fit than the wild type. Under duress from changing lighting due to clouds, we think it likely that all our mutations will make our organisms less fit, so that they are outcompeted by the wild type. For HSE, we could show this by comparing growth rates of the two organisms under the same conditions, or comparing growth directly through a competition assay. In such a situation, we can assay for our mutant by PCRing for the BioBrick prefix and suffix (this is the same method we would use to identify our organism in the environment, if needed). Conditions for this should ideally be as realistic as possible, mimicking the temperature and lighting we would expect in the environments where our organism could be accidentally released.<br />
</p><br />
<p><br />
Other evidence or information would need to be provided concerning DNA transfer. We would need to see which organisms our strain could potentially transfer genes to, and note the safety of those organisms (e.g. whether they are a toxin-producing genus like <i>Microcystis</i> or <i>Anabaena</i>). As the modifications we are making are chromosomal, our modified DNA is much less mobilisable than plasmid DNA. Literature on genetic transfer in cyanobacteria is not as dense as it is for <i>Escherichia coli</i>, for example, so it is more difficult to be certain about mutation rates (which might be important in determining the chance of inactivating a kill switch) or which species DNA can be transferred to. As with all cells, lysing of our bacteria will release DNA into the environment. In our risk assessment, we should give evidence that any genes we introduce occur naturally (or could occur naturally). In our case, several of our mutations also carry kanamycin resistance. In this case, we would would actually remove kanamycin resistance being using our organisms in an actual system. If we did leave in kanamycin resistance, we would again have to make it clear that kanamycin resistance genes occur naturally in the environment anyway. It should be noted that our organism does not produce toxins, and does not contribute to cyanobacterial blooms, so poses no obvious threat to the environment. It is also non-pathogenic so does not pose a threat to human health. It is possible that it could cause issues in an immunocompromised patient, though very unlikely. In our risk assessment, it would be best to look for cases of disease in immunocompromised patients being caused by <i>Synechocystis</i>, to show we are aware of potential issues.<br />
</p><br />
<br /><br />
<p><br />
For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.<br />
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<p><br />
Meeting experts in GMO safety and regulations, and consulting the appropriate legislation, has brought a number of key findings to light. Perhaps the most interesting is that current rules are not set up to cover a project like ours, that involves a container of GMMs outside and building, possibly on a person’s private property. As the Scientific Committees that in the EC are <a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">currently reviewing the risks of synthetic biology</a>, and that GMO-devices may become more common in the future, we may see regulations adapting more to cover these areas in the coming years.<br />
</p><br />
<p><br />
Although there are clearly areas where our panel counts as non-standard use, and so might be reclassified as higher risk than class 1, passing regulations may not be our biggest hurdle in getting our technology to the market. Our system complies with the safety measures needed, and our organism is of no or negligible risk to human safety or health, or the environment. Other obstacles that might be difficult to pass include how heavy our panels will be, and whether this will be a problem for transport or installing on rooftops, as having employees or members of the public on roofs would be a large safety risk in itself. The cost of submitting contained use applications to HSE also needs to be taken into account, and how the panels will be maintained without trained personnel on site. Furthermore, aspects of scaling up our technology also need to be worked out, such as what the optimal ratio of cyanobacteria-inoculated media to potassium ferricyanide is, and whether our cyanobacteria will survive long-term use in a photovoltaic cell. In addition, we do not know if people will be interested in continuing to pay for new media that must be added. By contrast, normal photovoltaic cells are one-off payments, even if they would be much more expensive than a cyanobacterial photovoltaic cell.<br />
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<p><br />
This is a bullet-point guide of what we’d need to do, step-by-step, to get our product to the market. It also provides a summary of our findings. It is by no means exhaustive though. We have focussed on the panels, rather than other aspects of our hypothetical business, and expect that many hurdles would magically appear to make life more difficult if we attempted to carry out our proposal.<br />
</p><br />
<p><br />
<ul><br />
<li>Get proof that our organisms our less fit (competition assay)</li><br />
<li>Get proof that our system is secure (leaks)</li><br />
<li>Carry out a risk assessment</li><br />
<li>Get advice from an expert person or panel on the risk assessment</li><br />
<li>Provide appropriate containment measures</li><br />
<li>Review the class of our work in respect to the containment measures</li><br />
<li>Draw up an emergency plan, inform the relevant personnel.</li><br />
<li>Submit an application to HSE, pay £452 for each site</li><br />
<li>Wait 10 days for acknowledgement of receipt</li><br />
<li>Commence work</li><br />
</ul><br />
<p><br />
Before any of this, it would be advisable to contact HSE to for advice on our proposition, however.<br />
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<p><br />
In this report we mainly referred to a few pieces of legislation, conversations we had with experts, and other sections of our wiki. As such, it made a lot more sense to link to all our resources as we went along, rather than using a Harvard or Vancouver style of referencing. However, we realise that it’s also convenient to have all the resources or references in one section. Here is a list of resources we used. If you’re hoping to review regulations on GMMs in the EU, this should be your starting point. <br />
</p><br />
<br /><br />
<p class="title"><i>Worldwide</i></p><br />
<p><br />
<b>The Cartagena Protocol</b> - UN-ratifed agreement for transborder GMO movement<br />
</p><br />
<br /><br />
<p class="title"><i>EU Directives</i></p><br />
<p><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009L0041&from=EN">Directive 2009/41/EC</a></b> - contained use <br /><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0018&from=EN">Directive 2001/18/EC</a></b> - deliberate release <br /><br />
<b>Directive 2000/54/EC</b> - risk classification of organisms <br /><br />
<b>Regulation EC 1946/2003</b> - transborder movement of GMOs. Implements the Cartagena Protocol <br /><br />
<b><a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">Opinion on Synthetic Biology</a></b> - first in a series to start reviewing risk in synthetic biology <br /><br />
For other EU legislation, start here <br /><br />
</p><br />
<br /><br />
<p class="title"><i>UK Regulations</i></p><br />
<p><br />
<b><a href="http://www.hse.gov.uk/pubns/books/l29.htm">Contained Use</a></b> <br /><br />
<b><a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/">SACGM Compendium of Guidance</a></b> <br /><br />
<b>Other UK rules</b> are mentioned <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">here</a> <br /><br />
<br />
</p><br />
<br /><br />
<p class="title"><i>Other</i></p><br />
<p><br />
We also found the <a href="http://biofuelpolicywatch.wordpress.com">BioFuel Policy Watch</a> blog and <a href="http://dglassassociates.wordpress.com">its associated blog</a> to be useful for general information. David Glass’s blog post on <a href="http://dglassassociates.wordpress.com/2013/09/22/regulation-of-industrial-use-of-algae-or-cyanobacteria-in-europe-part-1/">EU regulations for algae and cyanobacteria</a> is also a great starting point.<br />
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<p><br />
When we first asked the question, “can we put our bacteria on a roof?”, we didn’t envision giving such a detailed response. The proposition only reached its current form through repeated rounds of meetings with the students and supervisors, time spent reading EU and UK regulations and, most importantly, meetings with experts in biosafety at Reading. Gretta Roberts and Professor Jim Dunwell were very kind in giving up their time to answer all our questions, and we are very grateful for their input.<br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Human_PracticesTeam:Reading/Human Practices2014-10-17T22:46:03Z<p>Seafloor: </p>
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An important part of iGEM is thinking about the wider impact of your project. We considered whether it would be possible to set up our cyanobacterial solar panels on roofs at Reading or on people’s houses. This meant coming up with a <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">design for a larger photovoltaic cell</a>, considering the biosafety issues involved, and what regulatory challenges we would face.<br />
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<ol><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#levels">Levels of Regulation</a></li><br />
<li><a href="#eu">EU Regulations</a></li><br />
<ul><br />
<li><a href="#euintro">Introduction</a></li><br />
<li><a href="#eucontained">Contained Use</a></li><br />
<li><a href="#eudelib">Deliberate Release</a></li><br />
</ul><br />
<li><a href="#uk">UK Regulations</a></li><br />
<ul><br />
<li><a href="#ukintro">Introduction</a></li><br />
<li><a href="#ukcontained">Contained Use</a></li><br />
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<li><a href="#other">Other Regulations</a></li><br />
<li><a href="#safety">Biosafety</a></li><br />
<li><a href="#conc">Findings and Conclusions</a></li><br />
<li><a href="#road">The Roadmap</a></li><br />
<li><a href="#resources">Resources</a></li><br />
<li><a href="#acknowledgements">Acknowledgements</a></li><br />
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<td colspan="3"><h3 class="title" id="intro">Introduction</h3><br />
<p><br />
Creating a cyanobacterial photovoltaic cell and getting it installed on a roof at a university presents a number of challenges. The design of the system is the first aspect to consider. We look into design and cost of the parts on the Fuel Cell page. Then there are European Union (EU) and government regulations and, in the case of Reading, several boards and internal committees through which applications would have to pass. We consider these in this section. Each of these raises questions about biosafety, such as potential effects of the escape of our organism into surrounding environments. In addition to using our technology at our own university, we also considered commercialising the technology and installing it on people’s houses. This opens up a new realm of issues, such as getting our energy source classed as renewable according to the EU’s Renewable Energy Directive (RED), and getting permission for having GMOs on many distinct properties. These wider problems are also reviewed here.<br />
</p><br />
<p><br />
Many sections of our report will be applicable to other teams considering contained use of GMOs, and we hope future teams will benefit from our research. The EU section will be particularly relevant to other EU member states, as the EU regulations form the common minimum requirements for each country. In general, this page should guide teams considering biosafety issues associated with cyanobacteria; there are currently no reviews of biosafety in synthetic biology of cyanobacteria that we are aware of. We finish with a roadmap for those thinking about whether they could commercialise a genetically modified microorganism (GMM)-containing system, especially as a renewable fuel source.<br />
</p><br />
<p><br />
It should be noted that the report mainly refers to contained use of GMMs. Though our system is contained, parts of it could be considered to overlap with deliberate release. We have therefore focussed on regulations pertaining to contained use, but have referred to those on deliberate release where our system could potentially fall under its purview. Due to this relevance, and partly time due to time constraints, we have not exhaustively considered deliberate release or contained use categorised as class 2 or above.<br />
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<td colspan="3"><h3 class="title" id="levels">The Different Levels of Regulations</h3><br />
<p><br />
At the highest level, the Cartagena Protocol on Biosafety covers living modified organisms (LMOs) and their transport across borders. This is an international United Nations agreement that has been in place since 2003, and is implemented in the EU by <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>. Below that, the EU issues “directives” on genetically modified organisms (GMOs) that must be implemented by all EU member states. For contained use, only the state’s regulations need to be considered; there is no involvement at the EU level. For deliberate release, rules are much more complicated, involving notification of the European Commision (EC), and will not be covered here. In the UK, the EU directives are implemented by the Department for Environment, Food and Rural Affairs (DEFRA) and the Health and Safety Executive (HSE). Finally, at Reading we would have to pass at least 3 committees - including the sub-committee for biological safety, the project committee, and the environmental committee - in addition to getting approval from the building manager.<br />
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<td colspan="3"><h3 class="title" id="eu">EU Regulations</h3><br />
<p class="title" id="euintro"><i>Introduction to EU Regulations</i></p><br />
<p><br />
The two EU directives concerning our plans are the directive 2009/41/EC on contained use and the 2001/18/EC directive on deliberate release of GMOs. Of these, the contained use directive is probably most appropriate. The 2009/41/EC directive defines contained use as: “any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment”, in Article 2(c).<br />
</p><br />
<p><br />
By contrast, the 2001/18/EC directive defines deliberate release as: “any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment”, in Article 2(3).<br />
</p><br />
<p><br />
By comparing these, and reviewing <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the proposed implementation of our idea</a>, we can see that we would most likely fall under the contained use directive because of our suggested containment measures. Our technology will use all of the activities specified by contained use, and implement appropriate safety measures. Based on this, we shall chiefly address contained use regulations, but mention rules on environmental release that are significant.<br />
</p><br />
<br /><br />
<p class="title" id="eucontained"><i>EU: Contained Use</i></p><br />
<p><br />
In summary, class 1 contained use requires: <br /><br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment</li><br />
<li>classification of the risk of the GMM according to the assessment</li><br />
<li>appropriate containment</li><br />
<li>notification of the relevant authority</li><br />
<li>an emergency plan for accidental release</li><br />
</ul><br />
</p><br />
<p><br />
We shall explore each of these points in further detail.<br />
</p><br />
<p><br />
Article 4 defines one of the main requirements for contained use: for a risk assessment to be carried out (Article 4(2)), in accordance with the guidelines in Annex III, with the aim of classifying the GMM. The criteria include assessing the potential to cause disease, effect on the environment (Annex III (A1)), and harmful effects of the genetic material, recipient, donor, vector and final GMM (Annex III (A2)). The severity of these issues and the chance of them happening must also be analysed. As a non-pathogenic organism, capability of causing disease is not relevant to our organism. The most germaine section is in Annex III (A1), which lists considering “deleterious effects due to establishment or dissemination in the environment” and “deleterious effects due to the natural transfer of inserted genetic material to other organisms.” Annex III (B7) goes on to require that the proposed use of the microorganism be combined with the above assessment in assigning it to a class. “Non-standard operations” is mentioned as affecting classification (Annex III (B7 iii)); this term is ambiguous, but may encompass our suggestion of having GMMs on roofs of private properties. <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">Our drip-in/drip-out system</a>, with the filter needing autoclaving upon replacement, may also fall under non-standard use. This would need to be taken into consideration if attempting to use our technology commercially. From this, it is clear that our GMM belongs in class 1 (as defined in Article 4(3)), so requires level 1 containment measures, though any doubt raised from our “non-standard operations” might cause a more strict classification (Article 4(4)). For those wishing to classify their organism, Directive 2000/54/EC can be referred to, or classification systems of the specific country.<br />
</p><br />
<p><br />
The risk assessment has a particular focus on waste disposal (Article 4(5)), making our drip-out waste disposal system very important. As removing the filter from the panel might be a source of accidental release, careful planning of waste management should be high on our priority list. The final risk assessment must be given to the competent authority (Article 4(6)) - HSE in the case of the UK. Containment measures are defined in Annex IV.<br />
</p><br />
<p><br />
Article 6 poses a potential issue. It requires notifying authorities upon contained use at each new property. While this is reasonable for single use at the university, our idea of having installations on separate houses would mean giving the information listed in Annex V for each site, including the risk assessment, which individuals are responsible for supervising, and a description of the premises. This would mean that the risk assessment must be sufficiently comprehensive to envision all potential environments where an installation may be set up, and would mean extra administration work for our organisation. It may be that, in the future, EU directives would need to be altered in order to make GMM technologies like ours more easily available for public benefit. Deliberate release regulations already contain a separate section for commercial use, and contained use may be separated this way in the future too.<br />
</p><br />
<p><br />
For class 1 organisms, no further notification is needed before commencing with contained use (Article 7). For higher risk classes more information is needed; as our organism is only class 1 we will not consider this, but rules can be found in Articles 8 and 9.<br />
</p><br />
<p><br />
Further thought should be given to the minimum containment measures stipulated in Annex IV, and whether our system meets these conditions. There are different requirements given for different potential situations. Our proposition would most likely fall under “Containment and other protective measures for other activities” for the panel itself, but other containment procedures would need to be reviewed for labs where genetic modification is done and areas where GMMs are cultured. Almost all containment options for class 1 organisms in this category are optional according the Annex IV. It is likely that the level of containment assumed for this category is more severe than in our proposition (i.e. the EC has assumed all contained use will occur inside a building). As such, we should expect to see some or all of the containment measures to be required, rather than optional, for our project. <br />
</p><br />
<p><br />
Beyond the obvious physical containment, there are several possible containment measures we could be required to enforce. This includes control of aerosols during “addition of material to a closed system or transfer of material to another system”. This would include transferring cyanobacteria to the fuel cell, which would be done in a separate contained facility, and during the removal of waste or the waste filter <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">(see design section)</a>, which would have to be done on site. The latter is one of the biggest issues our project could face.<br />
</p><br />
<p><br />
Inactivation of waste containing GMMs is also listed as optional, but could potentially be required. Furthermore, the air leaving the system, assuming filter-sterilised air is bubbled through our panel, could have to be filtered to prevent or minimise release. The only point already required for class 1 work is that personnel wear protective clothing.<br />
</p><br />
<p><br />
According to Article 13, an emergency plan is required. This must be made available to the public, relevant bodies and authorities, and other concerned EU member states. The plan is required in case containment measures fail, leading to “serious danger, whether immediate or delayed, to humans outside the premises and/or to the environment”. No information is given on how extensive this plan should be, and no minimum requirements are given. Member state legislation must therefore be consulted for any rules on how the plan must be structured.<br />
</p><br />
<p><br />
Finally, it should be noted that member states may consult the public on the proposition if they decide it is relevant (Article 12).<br />
</p><br />
<br /><br />
<p class="title" id="eudelib"><i>EU: Deliberate Release</i></p><br />
<p><br />
Below is outlined some of the salient points from the EU directive on deliberate release. These may be useful to other teams reviewing regulations. The key points are:<br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment is required (Article 4; Annex III)</li><br />
<li>regulations are different for commercial and non-commercial use</li><br />
<li>for non-commercial GMO work (Part B):</li><br />
<ul><br />
<li>parties must give a risk assessment and monitor use and effects</li><br />
<li>the authority can tell the public</li><br />
<li>approved uses must be reported to the EU</li><br />
</ul><br />
<li>for commercial GMO work (Part C):</li><br />
<ul><br />
<li>parties must notify the relevant authority before placing the product on the market</li><br />
<li>putting it on the market is defined as making it available to 3rd parties</li><br />
<li>the authority produces an “assessment report”</li><br />
<li>this is given to the applicant, the EC and EU member states</li><br />
<li>decisions apply throughout the EU</li><br />
<li>the public must be notified</li><br />
<li>as of June 2014, <a href="http://ec.europa.eu/food/plant/gmo/legislation/future_rules_en.htm">member states can restrict or ban GMOs in their country</a> that have been approved for all states</li><br />
</ul><br />
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<td colspan="3"><h3 class="title" id="uk">UK Regulations</h3><br />
<p class="title" id="ukintro"><i>Introduction to UK Regulations</i></p><br />
<p><br />
<p><br />
The EU regulations are useful to us as they provide a baseline level of regulation we can expect if we try to implement our technology anywhere in the EU. In each EU member state, it is ultimately that country’s regulations which we must abide by. We will now consider what the regulations are like the UK. <br />
</p><br />
<p><br />
In general, they are slightly stricter than the basic EU regulations. The main regulations are <a href="http://www.hse.gov.uk/pubns/priced/l29.pdf">the HSE Contained Use regulations</a>, which are newly updated for 2014, and the accompanying SACGM Compendium of Guidance, which has yet to be updated to meet the new Contained Use document. Along with this, there are <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the regulations on deliberate release</a> from 1997, <a href="http://www.legislation.gov.uk/ukpga/1990/43/contents">section 108(1) of the Environment Protection Act </a> from 1990, and <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the Genetically Modified Organisms Regulations </a> (1996) that are related to GMOs. The latter three are all focussed on environmental release, so won’t be covered here. Regulations are upheld by the HSE and DEFRA. <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">The HSE website</a> can be consulted for all other regulations that might relate to the use of GMOs.<br />
</p><br />
<br /><br />
<p class="title" id="ukcontained"><i>UK Regulations: Contained Use</i></p><br />
<p><br />
The essential requirements for contained use in the UK are in the line with EU rules; we need to carry out a risk assessment and classify our organism, and notify the HSE before commencing GM work. So far we have only talked about the sites where the panels will be installed, but we will also have to consider regulations for handling, transport, work area decontamination, inactivation of GMMs and their disposal (including waste management). While the GMMs’ safety would need to be assessed by HSE, the system containing them, our panel, will need to be tested for leakages, with this evidence submitted to DEFRA.<br />
</p><br />
<p><br />
The definition given for contained use in Part 1, regulation 2 is “an activity in which organisms are genetically modified or in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers, or any combination of such barriers, are used to limit their contact with, and to provide a high level of protection for, humans and the environment”. Also in part 1, regulations 26 specifically tells us that commercial disposal of waste containing GMOs also falls under contained use. The contained use definition is similar to the EU definition, but is slightly more specific about what the containment measures must entail. In Part 1, paragraphs 24 and 45 give examples of what the barriers for our system might be expected to be. Physical could include a container, which would be the panel itself in our case. Chemical barriers may cover inactivation before waste disposal, and biological would include attenuating characteristics that debilitate the organism so that it is “rendered unable to survive outside of a specialised environment”. These barriers are discussed further in the safety page.<br />
</p><br />
<p><br />
The first requirement to consider is the risk assessment. For GMMs this is covered in Part 2, regulation 5, with more details on the assessment in Schedule 3 (Part 2). More emphasis is placed on risk to human and health and environment than in EU regulations. Regulation 5, paragraph 43, also answers questions we raised in the “EU: Contained Use” section about whether we could apply the same risk assessment to multiple sites with the same roof installation: “Where the contained use is identical at the multiple sites (eg in a clinical trial), the same risk assessment may apply to all the sites”. However it does point out that local changes in practices need to be taken into account. For us, practices would remain the same, but the surrounding environment may be different (e.g. there may be a pond at one property, where our organisms could theoretically survive). What the risk assessment must encompass is introduced in paragraph 44. In short, we must outline our plans, potential harmful effects, the chance of them occurring and their severity, and how we’ll deal with waste. These topics are covered in the safety page, where we look at each of our mutations; waste disposal is covered in <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the Fuel Cell page</a>.<br />
</p><br />
<br /><br />
<p><img align=centre src="https://static.igem.org/mediawiki/2014/7/77/Containment_lab_1.jpg" width="800px"></p><br />
<br /><br />
<p><br />
It is clear that the detail needed in the risk assessment is partly defined by how well-understood the microorganism and mutations are. Although our organism is clearly a non-pathogenic, non-hazardous class 1 organism, it is not as well understood as Escherichia coli K-12, for example, and the genes are not ones that are commonly used. They do not have a strong history of safe use, like green fluorescent protein (GFP). All our mutations have been done before, however, while measuring for different endpoints, so literature is available for reference on the effects of our mutations. From paragraphs 52 and 53, we know that the classification of the work changes to reflect the level of containment needed. When considering EU regulations, we were unsure of the extent of containment required, as our organism is class 1, but is used in unusual and potentially problematic environments. Under UK regulations, it appears our work could be reclassified to class 2 if we deem the containment measures for class 2 work to be desirable. This brings in a previously unforeseen hurdle: our work may be relabelled as higher than class 1 because of the containment measures needed on private properties with no trained personnel. We will continue to assume our work is class 1, but mention class 2 rules where appropriate.<br />
</p><br />
<p><br />
In summary, for the risk assessment we must identify hazards, assign appropriate containment measures, then reclassify our work based on these (if necessary). These instructions are similar to, but more detailed than, those for EU member states in general.<br />
</p><br />
<p><br />
According to regulation 8, we need to obtain advice from a person or committee on the risk assessment. If our organism were reclassified as class 2, this would have to be a biological safety committee. At Reading this would not be an issue, as there is already a committee from which we could obtain advice. If the classification remained as class 1, our meeting with Gretta Roberts and Professor Jim Dunwell, who have advised us with regards to regulations and safety, should be sufficient.<br />
</p><br />
<p><br />
Regulation 9 requires notification of premises to be employed for contained use. We must first submit the information in Schedule 5, then wait 10 days for a response. A single notification may include more than 1 premises in situations where more than one premise is owned by the same company, so it’s possible that multiple sites could be asked about at once. This would reduce the cost of notifying HSE, which is currently <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£472 for class 1</a> and <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£943 for class 2</a>. However, if our organism still falls under class 1, we only need to submit a summary of the risk assessment, details on waste management and the advice we received during the risk assessment, and confirmation that relevant authorities will be notified of the emergency plan. The rest of the information needed is basic details like the address of the premises. For class 2 work, regulation 10 should be consulted.<br />
</p><br />
<p><br />
Part 3 outlines practices that must occur for contained use. Regulations 18, 19, 21 and 22 are all relevant to different stages of our plans. Sections relevant specifically to the panels include paragraphs 107-109, which tell us that containment measures must be tested; this may involve checking each panel for defects or leaking before deployment, and possibly visiting sites at intervals to check they are still functioning correctly (paragraph 108). This could mean simply looking over the panel for any cracks or leakages to check for physical containment. Checking that biological containment is still in place, for example by checking that cells cannot directly transfer or uptake DNA after the pilT1 mutation, may involve taking a sample from panels. This itself means removing liquid containing GMMs and transporting it back to the lab for testing transformation efficiency; we would have to ensure that taking any liquid samples would not have any risks of accidental release. It is unlikely, but possible, that we could be required to check for our GMM in the surrounding environment to ensure there had been no release (paragraph 111).<br />
</p><br />
<p><br />
Regulation 19 specifically covers containment measures for GMMs. Containment measures must be reviewed “at regular intervals” (paragraphs 128 and 129), which must occur more frequently for non-standard work. According to paragraph 134, our organism does not fit the criteria for class 1 work that does not require waste inactivation, because our organism does not contain “multiple disabling mutations”, so waste from our panel must be inactivated. According to 135, it is acceptable to take the filter to another location and autoclave it, assuming steps are taken to make sure storage and transport are safe, and that the process is effective at inactivating our GMM.<br />
</p><br />
<p><br />
We must follow Schedule 8, Part 2, Table 2 for containment measures. As with our assessment of EU containment requirements, we have chosen the “other” section, as this seems most appropriate. Here the only absolute requirement is that personnel must wear work clothing. Measures that might be required, if deemed so by the risk assessment, include: physical separation, control of aerosols (as discussed in the EU contained use section), inactivation of waste or removed fluid, and control for spillage. The last of these is the only one not fully considered so far in our design.<br />
</p><br />
<p><br />
Emergency plans are discussed in regulation 21. According to paragraph 139, however, “an emergency plan should only be prepared for work with organisms that pose the highest hazards to humans or the environment.” Although our organism is low hazard, the risk is perhaps higher because the GMMs are not contained in a facility. As only hazard is mentioned, it is possible that we would not have to draw up an emergency plan (for our organism the hazard is low, but the risk slightly higher as it is not contained in a building). Furthermore, only those on the premises would be exposed to our non-dangerous GMM, but paragraph 139 only requires an emergency plan when the health or safety of those outside the premises is in danger. This probably assumes the work is a designated building with trained personnel on site, though. Given this, and our non-standard plans, it would be prudent to anticipate an emergency plan being required. If this were the case, it must be submitted along with our contained used application to HSE (paragraph 140). Paragraph 141 covers the requirement for an emergency plan; those “on the site affected by the plan” should know the plan, so residents in houses where a panel is installed, or building managers for a university building, would have to be familiar with the plan (paragraph 142). The plan must also be publicly available (paragraph 143), as we know from the EU requirements. Although we have primarily talked about the risk to the environment of our GMMs, we should also consider the risks of releasing potassium ferricyanide to the environment (a part of our solar panel), like how much would be released, any risks to human health or safety, and any risks to the environment.<br />
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<td colspan="3"><h3 class="title" id="other">Other Regulations</h3><br />
<p><br />
We’ve covered the main EU and UK regulations regarding the installation of our actual panels, but there are many other regulations relevant to our plans. Contained use regulations are the main ones, which would cover the genetic modification of our organisms in a lab, the transport of our organisms to a facility where they can be grown up, that facility itself, transport to houses or the university, and the property where the panel is installed. There are other regulations that could come into play, however. This includes rules on the <a href="http://www.hse.gov.uk/cdg/introduction.htm">carriage of dangerous goods</a>, which might include our GMMs or potassium ferricyanide. Furthermore, transport regulations for crossing borders in the EU, which might occur if we export cultivation of our GMMs to another country, would mean we must adhere to EU GMO border rules in <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>, which implements the Cartagena Protocol.<br />
</p><br />
<p><br />
At Reading, we would also have to submit an application to at least 3 committees before getting our proposal approved, and speak to the building manager for each building we would want to get our panel put on. Although the committees sound like more regulatory hurdles, the university safety officers would contact HSE for us, and we would supply all our information to the safety officers, making the process much easier. Furthermore, the biological safety committee would be the committee we would contact for the expert advice required when carrying out a risk assessment, and all the buildings at the university count as one private property, for which only one application to HSE needs to be made.<br />
</p><br />
<p><br />
Commercialisation of our product would mean selling it as a new source of renewable energy. Renewable fuel sources are subject to other rules in the EU, which stipulate criteria that must be met for a source to be labelled as “renewable”. The Fuel Quality Directive (FQD) is relevant to renewable fuels for transport, such as biofuels, and the Renewable Energy Directive (RED) is pertinent to other energy sources that wish to be labelled as renewable; ours could fall under the latter. The requirements in RED are essentially requirements for EU member states, but in order for the standards to be met, it is individual companies that ultimately must comply. The RED is enforced by the European Commission Directorate General for Energy. The requirements include showing a reduction in greenhouse gas emissions over the course of the fuel’s production, and using life cycle analysis (LCA) methods to calculate the “carbon intensity” of our energy source. Our “sustainability analysis” must encompass other features beyond an LCA. To meet the specifications in the RED, we must check 12 independent factors of our energy source (including its production and transportation), and have this verified by a third party. The analysis method must be approved by the EU.<br />
</p><br />
<p><br />
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<td colspan="3"><h3 class="title" id="safety">Biosafety</h3><br />
<p><br />
We have now reviewed the safety requirements at the EU and UK levels. The exact steps for the UK risk assessment are outline in Section 3, part 2, of the Contained Use regulations. We will not go through all of them here, but we can comment on what is required. First, it is clear that we will have to prove our organisms are less fit than the wild type. Under duress from changing lighting due to clouds, we think it likely that all our mutations will make our organisms less fit, so that they are outcompeted by the wild type. For HSE, we could show this by comparing growth rates of the two organisms under the same conditions, or comparing growth directly through a competition assay. In such a situation, we can assay for our mutant by PCRing for the BioBrick prefix and suffix (this is the same method we would use to identify our organism in the environment, if needed). Conditions for this should ideally be as realistic as possible, mimicking the temperature and lighting we would expect in the environments where our organism could be accidentally released.<br />
</p><br />
<p><br />
Other evidence or information would need to be provided concerning DNA transfer. We would need to see which organisms our strain could potentially transfer genes to, and note the safety of those organisms (e.g. whether they are a toxin-producing genus like <i>Microcystis</i> or <i>Anabaena</i>). As the modifications we are making are chromosomal, our modified DNA is much less mobilisable than plasmid DNA. Literature on genetic transfer in cyanobacteria is not as dense as it is for <i>Escherichia coli</i>, for example, so it is more difficult to be certain about mutation rates (which might be important in determining the chance of inactivating a kill switch) or which species DNA can be transferred to. As with all cells, lysing of our bacteria will release DNA into the environment. In our risk assessment, we should give evidence that any genes we introduce occur naturally (or could occur naturally). In our case, several of our mutations also carry kanamycin resistance. In this case, we would would actually remove kanamycin resistance being using our organisms in an actual system. If we did leave in kanamycin resistance, we would again have to make it clear that kanamycin resistance genes occur naturally in the environment anyway. It should be noted that our organism does not produce toxins, and does not contribute to cyanobacterial blooms, so poses no obvious threat to the environment. It is also non-pathogenic so does not pose a threat to human health. It is possible that it could cause issues in an immunocompromised patient, though very unlikely. In our risk assessment, it would be best to look for cases of disease in immunocompromised patients being caused by <i>Synechocystis</i>, to show we are aware of potential issues.<br />
</p><br />
<br /><br />
<p><br />
For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.<br />
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<p><br />
Meeting experts in GMO safety and regulations, and consulting the appropriate legislation, has brought a number of key findings to light. Perhaps the most interesting is that current rules are not set up to cover a project like ours, that involves a container of GMMs outside and building, possibly on a person’s private property. As the Scientific Committees that in the EC are <a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">currently reviewing the risks of synthetic biology</a>, and that GMO-devices may become more common in the future, we may see regulations adapting more to cover these areas in the coming years.<br />
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<p><br />
Although there are clearly areas where our panel counts as non-standard use, and so might be reclassified as higher risk than class 1, passing regulations may not be our biggest hurdle in getting our technology to the market. Our system complies with the safety measures needed, and our organism is of no or negligible risk to human safety or health, or the environment. Other obstacles that might be difficult to pass include how heavy our panels will be, and whether this will be a problem for transport or installing on rooftops, as having employees or members of the public on roofs would be a large safety risk in itself. The cost of submitting contained use applications to HSE also needs to be taken into account, and how the panels will be maintained without trained personnel on site. Furthermore, aspects of scaling up our technology also need to be worked out, such as what the optimal ratio of cyanobacteria-inoculated media to potassium ferricyanide is, and whether our cyanobacteria will survive long-term use in a photovoltaic cell. In addition, we do not know if people will be interested in continuing to pay for new media that must be added. By contrast, normal photovoltaic cells are one-off payments, even if they would be much more expensive than a cyanobacterial photovoltaic cell.<br />
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<p><br />
This is a bullet-point guide of what we’d need to do, step-by-step, to get our product to the market. It also provides a summary of our findings. It is by no means exhaustive though. We have focussed on the panels, rather than other aspects of our hypothetical business, and expect that many hurdles would magically appear to make life more difficult if we attempted to carry out our proposal.<br />
</p><br />
<p><br />
<ul><br />
<li>Get proof that our organisms our less fit (competition assay)</li><br />
<li>Get proof that our system is secure (leaks)</li><br />
<li>Carry out a risk assessment</li><br />
<li>Get advice from an expert person or panel on the risk assessment</li><br />
<li>Provide appropriate containment measures</li><br />
<li>Review the class of our work in respect to the containment measures</li><br />
<li>Draw up an emergency plan, inform the relevant personnel.</li><br />
<li>Submit an application to HSE, pay £452 for each site</li><br />
<li>Wait 10 days for acknowledgement of receipt</li><br />
<li>Commence work</li><br />
</ul><br />
<p><br />
Before any of this, it would be advisable to contact HSE to for advice on our proposition, however.<br />
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<p><br />
In this report we mainly referred to a few pieces of legislation, conversations we had with experts, and other sections of our wiki. As such, it made a lot more sense to link to all our resources as we went along, rather than using a Harvard or Vancouver style of referencing. However, we realise that it’s also convenient to have all the resources or references in one section. Here is a list of resources we used. If you’re hoping to review regulations on GMMs in the EU, this should be your starting point. <br />
</p><br />
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<p class="title"><i>Worldwide</i></p><br />
<p><br />
<b>The Cartagena Protocol</b> - UN-ratifed agreement for transborder GMO movement<br />
</p><br />
<br /><br />
<p class="title"><i>EU Directives</i></p><br />
<p><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009L0041&from=EN">Directive 2009/41/EC</a></b> - contained use <br /><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0018&from=EN">Directive 2001/18/EC</a></b> - deliberate release <br /><br />
<b>Directive 2000/54/EC</b> - risk classification of organisms <br /><br />
<b>Regulation EC 1946/2003</b> - transborder movement of GMOs. Implements the Cartagena Protocol <br /><br />
<b><a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">Opinion on Synthetic Biology</a></b> - first in a series to start reviewing risk in synthetic biology <br /><br />
For other EU legislation, start here <br /><br />
</p><br />
<br /><br />
<p class="title"><i>UK Regulations</i></p><br />
<p><br />
<b><a href="http://www.hse.gov.uk/pubns/books/l29.htm">Contained Use</a></b> <br /><br />
<b><a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/">SACGM Compendium of Guidance</a></b> <br /><br />
<b>Other UK rules</b> are mentioned <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">here</a> <br /><br />
<br />
</p><br />
<br /><br />
<p class="title"><i>Other</i></p><br />
<p><br />
We also found the <a href="http://biofuelpolicywatch.wordpress.com">BioFuel Policy Watch</a> blog and <a href="http://dglassassociates.wordpress.com">its associated blog</a> to be useful for general information. David Glass’s blog post on <a href="http://dglassassociates.wordpress.com/2013/09/22/regulation-of-industrial-use-of-algae-or-cyanobacteria-in-europe-part-1/">EU regulations for algae and cyanobacteria</a> is also a great starting point.<br />
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<p><br />
When we first asked the question, “can we put our bacteria on a roof?”, we didn’t envision giving such a detailed response. The proposition only reached its current form through repeated rounds of meetings with the students and supervisors, time spent reading EU and UK regulations and, most importantly, meetings with experts in biosafety at Reading. Gretta Roberts and Professor Jim Dunwell were very kind in giving up their time to answer all our questions, and we are very grateful for their input.<br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Human_PracticesTeam:Reading/Human Practices2014-10-17T22:12:59Z<p>Seafloor: </p>
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An important part of iGEM is thinking about the wider impact of your project. We considered whether it would be possible to set up our cyanobacterial solar panels on roofs at Reading or on people’s houses. This meant coming up with a <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">design for a larger photovoltaic cell</a>, considering the biosafety issues involved, and what regulatory challenges we would face.<br />
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<p><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/3/30/Cyano_cultures.jpg" width="800px"></p><br />
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<ol><br />
<li><a href="#summary">Summary</a></li><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#levels">Levels of Regulation</a></li><br />
<li><a href="#eu">EU Regulations</a></li><br />
<ul><br />
<li><a href="#euintro">Introduction</a></li><br />
<li><a href="#eucontained">Contained Use</a></li><br />
<li><a href="#eudelib">Deliberate Release</a></li><br />
</ul><br />
<li><a href="#uk">UK Regulations</a></li><br />
<ul><br />
<li><a href="#ukintro">Introduction</a></li><br />
<li><a href="#ukcontained">Contained Use</a></li><br />
</ul><br />
<li><a href="#other">Other Regulations</a></li><br />
<li><a href="#safety">Biosafety</a></li><br />
<li><a href="#conc">Findings and Conclusions</a></li><br />
<li><a href="#road">The Roadmap</a></li><br />
<li><a href="#resources">Resources</a></li><br />
<li><a href="#acknowledgements">Acknowledgements</a></li><br />
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<p><br />
Creating a cyanobacterial photovoltaic cell and getting it installed on a roof at a university presents a number of challenges. The design of the system is the first aspect to consider. We look into design and cost of the parts on the Fuel Cell page. Then there are European Union (EU) and government regulations and, in the case of Reading, several boards and internal committees through which applications would have to pass. We consider these in this section. Each of these raises questions about biosafety, such as potential effects of the escape of our organism into surrounding environments. In addition to using our technology at our own university, we also considered commercialising the technology and installing it on people’s houses. This opens up a new realm of issues, such as getting our energy source classed as renewable according to the EU’s Renewable Energy Directive (RED), and getting permission for having GMOs on many distinct properties. These wider problems are also reviewed here.<br />
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<p><br />
Many sections of our report will be applicable to other teams considering contained use of GMOs, and we hope future teams will benefit from our research. The EU section will be particularly relevant to other EU member states, as the EU regulations form the common minimum requirements for each country. In general, this page should guide teams considering biosafety issues associated with cyanobacteria; there are currently no reviews of biosafety in synthetic biology of cyanobacteria that we are aware of. We finish with a roadmap for those thinking about whether they could commercialise a genetically modified microorganism (GMM)-containing system, especially as a renewable fuel source.<br />
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<p><br />
It should be noted that the report mainly refers to contained use of GMMs. Though our system is contained, parts of it could be considered to overlap with deliberate release. We have therefore focussed on regulations pertaining to contained use, but have referred to those on deliberate release where our system could potentially fall under its purview. Due to this relevance, and partly time due to time constraints, we have not exhaustively considered deliberate release or contained use categorised as class 2 or above.<br />
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<p><br />
At the highest level, the Cartagena Protocol on Biosafety covers living modified organisms (LMOs) and their transport across borders. This is an international United Nations agreement that has been in place since 2003, and is implemented in the EU by <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>. Below that, the EU issues “directives” on genetically modified organisms (GMOs) that must be implemented by all EU member states. For contained use, only the state’s regulations need to be considered; there is no involvement at the EU level. For deliberate release, rules are much more complicated, involving notification of the European Commision (EC), and will not be covered here. In the UK, the EU directives are implemented by the Department for Environment, Food and Rural Affairs (DEFRA) and the Health and Safety Executive (HSE). Finally, at Reading we would have to pass at least 3 committees - including the sub-committee for biological safety, the project committee, and the environmental committee - in addition to getting approval from the building manager.<br />
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<p class="title" id="euintro"><i>Introduction to EU Regulations</i></p><br />
<p><br />
The two EU directives concerning our plans are the directive 2009/41/EC on contained use and the 2001/18/EC directive on deliberate release of GMOs. Of these, the contained use directive is probably most appropriate. The 2009/41/EC directive defines contained use as: “any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment”, in Article 2(c).<br />
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<p><br />
By contrast, the 2001/18/EC directive defines deliberate release as: “any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment”, in Article 2(3).<br />
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<p><br />
By comparing these, and reviewing <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the proposed implementation of our idea</a>, we can see that we would most likely fall under the contained use directive because of our suggested containment measures. Our technology will use all of the activities specified by contained use, and implement appropriate safety measures. Based on this, we shall chiefly address contained use regulations, but mention rules on environmental release that are significant.<br />
</p><br />
<br /><br />
<p class="title" id="eucontained"><i>EU: Contained Use</i></p><br />
<p><br />
In summary, class 1 contained use requires: <br /><br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment</li><br />
<li>classification of the risk of the GMM according to the assessment</li><br />
<li>appropriate containment</li><br />
<li>notification of the relevant authority</li><br />
<li>an emergency plan for accidental release</li><br />
</ul><br />
</p><br />
<p><br />
We shall explore each of these points in further detail.<br />
</p><br />
<p><br />
Article 4 defines one of the main requirements for contained use: for a risk assessment to be carried out (Article 4(2)), in accordance with the guidelines in Annex III, with the aim of classifying the GMM. The criteria include assessing the potential to cause disease, effect on the environment (Annex III (A1)), and harmful effects of the genetic material, recipient, donor, vector and final GMM (Annex III (A2)). The severity of these issues and the chance of them happening must also be analysed. As a non-pathogenic organism, capability of causing disease is not relevant to our organism. The most germaine section is in Annex III (A1), which lists considering “deleterious effects due to establishment or dissemination in the environment” and “deleterious effects due to the natural transfer of inserted genetic material to other organisms.” Annex III (B7) goes on to require that the proposed use of the microorganism be combined with the above assessment in assigning it to a class. “Non-standard operations” is mentioned as affecting classification (Annex III (B7 iii)); this term is ambiguous, but may encompass our suggestion of having GMMs on roofs of private properties. <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">Our drip-in/drip-out system</a>, with the filter needing autoclaving upon replacement, may also fall under non-standard use. This would need to be taken into consideration if attempting to use our technology commercially. From this, it is clear that our GMM belongs in class 1 (as defined in Article 4(3)), so requires level 1 containment measures, though any doubt raised from our “non-standard operations” might cause a more strict classification (Article 4(4)). For those wishing to classify their organism, Directive 2000/54/EC can be referred to, or classification systems of the specific country.<br />
</p><br />
<p><br />
The risk assessment has a particular focus on waste disposal (Article 4(5)), making our drip-out waste disposal system very important. As removing the filter from the panel might be a source of accidental release, careful planning of waste management should be high on our priority list. The final risk assessment must be given to the competent authority (Article 4(6)) - HSE in the case of the UK. Containment measures are defined in Annex IV.<br />
</p><br />
<p><br />
Article 6 poses a potential issue. It requires notifying authorities upon contained use at each new property. While this is reasonable for single use at the university, our idea of having installations on separate houses would mean giving the information listed in Annex V for each site, including the risk assessment, which individuals are responsible for supervising, and a description of the premises. This would mean that the risk assessment must be sufficiently comprehensive to envision all potential environments where an installation may be set up, and would mean extra administration work for our organisation. It may be that, in the future, EU directives would need to be altered in order to make GMM technologies like ours more easily available for public benefit. Deliberate release regulations already contain a separate section for commercial use, and contained use may be separated this way in the future too.<br />
</p><br />
<p><br />
For class 1 organisms, no further notification is needed before commencing with contained use (Article 7). For higher risk classes more information is needed; as our organism is only class 1 we will not consider this, but rules can be found in Articles 8 and 9.<br />
</p><br />
<p><br />
Further thought should be given to the minimum containment measures stipulated in Annex IV, and whether our system meets these conditions. There are different requirements given for different potential situations. Our proposition would most likely fall under “Containment and other protective measures for other activities” for the panel itself, but other containment procedures would need to be reviewed for labs where genetic modification is done and areas where GMMs are cultured. Almost all containment options for class 1 organisms in this category are optional according the Annex IV. It is likely that the level of containment assumed for this category is more severe than in our proposition (i.e. the EC has assumed all contained use will occur inside a building). As such, we should expect to see some or all of the containment measures to be required, rather than optional, for our project. <br />
</p><br />
<p><br />
Beyond the obvious physical containment, there are several possible containment measures we could be required to enforce. This includes control of aerosols during “addition of material to a closed system or transfer of material to another system”. This would include transferring cyanobacteria to the fuel cell, which would be done in a separate contained facility, and during the removal of waste or the waste filter <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">(see design section)</a>, which would have to be done on site. The latter is one of the biggest issues our project could face.<br />
</p><br />
<p><br />
Inactivation of waste containing GMMs is also listed as optional, but could potentially be required. Furthermore, the air leaving the system, assuming filter-sterilised air is bubbled through our panel, could have to be filtered to prevent or minimise release. The only point already required for class 1 work is that personnel wear protective clothing.<br />
</p><br />
<p><br />
According to Article 13, an emergency plan is required. This must be made available to the public, relevant bodies and authorities, and other concerned EU member states. The plan is required in case containment measures fail, leading to “serious danger, whether immediate or delayed, to humans outside the premises and/or to the environment”. No information is given on how extensive this plan should be, and no minimum requirements are given. Member state legislation must therefore be consulted for any rules on how the plan must be structured.<br />
</p><br />
<p><br />
Finally, it should be noted that member states may consult the public on the proposition if they decide it is relevant (Article 12).<br />
</p><br />
<br /><br />
<p class="title" id="eudelib"><i>EU: Deliberate Release</i></p><br />
<p><br />
Below is outlined some of the salient points from the EU directive on deliberate release. These may be useful to other teams reviewing regulations. The key points are:<br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment is required (Article 4; Annex III)</li><br />
<li>regulations are different for commercial and non-commercial use</li><br />
<li>for non-commercial GMO work (Part B):</li><br />
<ul><br />
<li>parties must give a risk assessment and monitor use and effects</li><br />
<li>the authority can tell the public</li><br />
<li>approved uses must be reported to the EU</li><br />
</ul><br />
<li>for commercial GMO work (Part C):</li><br />
<ul><br />
<li>parties must notify the relevant authority before placing the product on the market</li><br />
<li>putting it on the market is defined as making it available to 3rd parties</li><br />
<li>the authority produces an “assessment report”</li><br />
<li>this is given to the applicant, the EC and EU member states</li><br />
<li>decisions apply throughout the EU</li><br />
<li>the public must be notified</li><br />
<li>as of June 2014, <a href="http://ec.europa.eu/food/plant/gmo/legislation/future_rules_en.htm">member states can restrict or ban GMOs in their country</a> that have been approved for all states</li><br />
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<p class="title" id="ukintro"><i>Introduction to UK Regulations</i></p><br />
<p><br />
<p><br />
The EU regulations are useful to us as they provide a baseline level of regulation we can expect if we try to implement our technology anywhere in the EU. In each EU member state, it is ultimately that country’s regulations which we must abide by. We will now consider what the regulations are like the UK. <br />
</p><br />
<p><br />
In general, they are slightly stricter than the basic EU regulations. The main regulations are <a href="http://www.hse.gov.uk/pubns/priced/l29.pdf">the HSE Contained Use regulations</a>, which are newly updated for 2014, and the accompanying SACGM Compendium of Guidance, which has yet to be updated to meet the new Contained Use document. Along with this, there are <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the regulations on deliberate release</a> from 1997, <a href="http://www.legislation.gov.uk/ukpga/1990/43/contents">section 108(1) of the Environment Protection Act </a> from 1990, and <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the Genetically Modified Organisms Regulations </a> (1996) that are related to GMOs. The latter three are all focussed on environmental release, so won’t be covered here. Regulations are upheld by the HSE and DEFRA. <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">The HSE website</a> can be consulted for all other regulations that might relate to the use of GMOs.<br />
</p><br />
<br /><br />
<p class="title" id="ukcontained"><i>UK Regulations: Contained Use</i></p><br />
<p><br />
The essential requirements for contained use in the UK are in the line with EU rules; we need to carry out a risk assessment and classify our organism, and notify the HSE before commencing GM work. So far we have only talked about the sites where the panels will be installed, but we will also have to consider regulations for handling, transport, work area decontamination, inactivation of GMMs and their disposal (including waste management). While the GMMs’ safety would need to be assessed by HSE, the system containing them, our panel, will need to be tested for leakages, with this evidence submitted to DEFRA.<br />
</p><br />
<p><br />
The definition given for contained use in Part 1, regulation 2 is “an activity in which organisms are genetically modified or in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers, or any combination of such barriers, are used to limit their contact with, and to provide a high level of protection for, humans and the environment”. Also in part 1, regulations 26 specifically tells us that commercial disposal of waste containing GMOs also falls under contained use. The contained use definition is similar to the EU definition, but is slightly more specific about what the containment measures must entail. In Part 1, paragraphs 24 and 45 give examples of what the barriers for our system might be expected to be. Physical could include a container, which would be the panel itself in our case. Chemical barriers may cover inactivation before waste disposal, and biological would include attenuating characteristics that debilitate the organism so that it is “rendered unable to survive outside of a specialised environment”. These barriers are discussed further in the safety page.<br />
</p><br />
<p><br />
The first requirement to consider is the risk assessment. For GMMs this is covered in Part 2, regulation 5, with more details on the assessment in Schedule 3 (Part 2). More emphasis is placed on risk to human and health and environment than in EU regulations. Regulation 5, paragraph 43, also answers questions we raised in the “EU: Contained Use” section about whether we could apply the same risk assessment to multiple sites with the same roof installation: “Where the contained use is identical at the multiple sites (eg in a clinical trial), the same risk assessment may apply to all the sites”. However it does point out that local changes in practices need to be taken into account. For us, practices would remain the same, but the surrounding environment may be different (e.g. there may be a pond at one property, where our organisms could theoretically survive). What the risk assessment must encompass is introduced in paragraph 44. In short, we must outline our plans, potential harmful effects, the chance of them occurring and their severity, and how we’ll deal with waste. These topics are covered in the safety page, where we look at each of our mutations; waste disposal is covered in <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the Fuel Cell page</a>.<br />
</p><br />
<p><br />
It is clear that the detail needed in the risk assessment is partly defined by how well-understood the microorganism and mutations are. Although our organism is clearly a non-pathogenic, non-hazardous class 1 organism, it is not as well understood as Escherichia coli K-12, for example, and the genes are not ones that are commonly used. They do not have a strong history of safe use, like green fluorescent protein (GFP). All our mutations have been done before, however, while measuring for different endpoints, so literature is available for reference on the effects of our mutations. From paragraphs 52 and 53, we know that the classification of the work changes to reflect the level of containment needed. When considering EU regulations, we were unsure of the extent of containment required, as our organism is class 1, but is used in unusual and potentially problematic environments. Under UK regulations, it appears our work could be reclassified to class 2 if we deem the containment measures for class 2 work to be desirable. This brings in a previously unforeseen hurdle: our work may be relabelled as higher than class 1 because of the containment measures needed on private properties with no trained personnel. We will continue to assume our work is class 1, but mention class 2 rules where appropriate.<br />
</p><br />
<p><br />
In summary, for the risk assessment we must identify hazards, assign appropriate containment measures, then reclassify our work based on these (if necessary). These instructions are similar to, but more detailed than, those for EU member states in general.<br />
</p><br />
<p><br />
According to regulation 8, we need to obtain advice from a person or committee on the risk assessment. If our organism were reclassified as class 2, this would have to be a biological safety committee. At Reading this would not be an issue, as there is already a committee from which we could obtain advice. If the classification remained as class 1, our meeting with Gretta Roberts and Professor Jim Dunwell, who have advised us with regards to regulations and safety, should be sufficient.<br />
</p><br />
<p><br />
Regulation 9 requires notification of premises to be employed for contained use. We must first submit the information in Schedule 5, then wait 10 days for a response. A single notification may include more than 1 premises in situations where more than one premise is owned by the same company, so it’s possible that multiple sites could be asked about at once. This would reduce the cost of notifying HSE, which is currently <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£472 for class 1</a> and <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£943 for class 2</a>. However, if our organism still falls under class 1, we only need to submit a summary of the risk assessment, details on waste management and the advice we received during the risk assessment, and confirmation that relevant authorities will be notified of the emergency plan. The rest of the information needed is basic details like the address of the premises. For class 2 work, regulation 10 should be consulted.<br />
</p><br />
<p><br />
Part 3 outlines practices that must occur for contained use. Regulations 18, 19, 21 and 22 are all relevant to different stages of our plans. Sections relevant specifically to the panels include paragraphs 107-109, which tell us that containment measures must be tested; this may involve checking each panel for defects or leaking before deployment, and possibly visiting sites at intervals to check they are still functioning correctly (paragraph 108). This could mean simply looking over the panel for any cracks or leakages to check for physical containment. Checking that biological containment is still in place, for example by checking that cells cannot directly transfer or uptake DNA after the pilT1 mutation, may involve taking a sample from panels. This itself means removing liquid containing GMMs and transporting it back to the lab for testing transformation efficiency; we would have to ensure that taking any liquid samples would not have any risks of accidental release. It is unlikely, but possible, that we could be required to check for our GMM in the surrounding environment to ensure there had been no release (paragraph 111).<br />
</p><br />
<p><br />
Regulation 19 specifically covers containment measures for GMMs. Containment measures must be reviewed “at regular intervals” (paragraphs 128 and 129), which must occur more frequently for non-standard work. According to paragraph 134, our organism does not fit the criteria for class 1 work that does not require waste inactivation, because our organism does not contain “multiple disabling mutations”, so waste from our panel must be inactivated. According to 135, it is acceptable to take the filter to another location and autoclave it, assuming steps are taken to make sure storage and transport are safe, and that the process is effective at inactivating our GMM.<br />
</p><br />
<p><br />
We must follow Schedule 8, Part 2, Table 2 for containment measures. As with our assessment of EU containment requirements, we have chosen the “other” section, as this seems most appropriate. Here the only absolute requirement is that personnel must wear work clothing. Measures that might be required, if deemed so by the risk assessment, include: physical separation, control of aerosols (as discussed in the EU contained use section), inactivation of waste or removed fluid, and control for spillage. The last of these is the only one not fully considered so far in our design.<br />
</p><br />
<p><br />
Emergency plans are discussed in regulation 21. According to paragraph 139, however, “an emergency plan should only be prepared for work with organisms that pose the highest hazards to humans or the environment.” Although our organism is low hazard, the risk is perhaps higher because the GMMs are not contained in a facility. As only hazard is mentioned, it is possible that we would not have to draw up an emergency plan (for our organism the hazard is low, but the risk slightly higher as it is not contained in a building). Furthermore, only those on the premises would be exposed to our non-dangerous GMM, but paragraph 139 only requires an emergency plan when the health or safety of those outside the premises is in danger. This probably assumes the work is a designated building with trained personnel on site, though. Given this, and our non-standard plans, it would be prudent to anticipate an emergency plan being required. If this were the case, it must be submitted along with our contained used application to HSE (paragraph 140). Paragraph 141 covers the requirement for an emergency plan; those “on the site affected by the plan” should know the plan, so residents in houses where a panel is installed, or building managers for a university building, would have to be familiar with the plan (paragraph 142). The plan must also be publicly available (paragraph 143), as we know from the EU requirements. Although we have primarily talked about the risk to the environment of our GMMs, we should also consider the risks of releasing potassium ferricyanide to the environment (a part of our solar panel), like how much would be released, any risks to human health or safety, and any risks to the environment.<br />
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<p><br />
We’ve covered the main EU and UK regulations regarding the installation of our actual panels, but there are many other regulations relevant to our plans. Contained use regulations are the main ones, which would cover the genetic modification of our organisms in a lab, the transport of our organisms to a facility where they can be grown up, that facility itself, transport to houses or the university, and the property where the panel is installed. There are other regulations that could come into play, however. This includes rules on the <a href="http://www.hse.gov.uk/cdg/introduction.htm">carriage of dangerous goods</a>, which might include our GMMs or potassium ferricyanide. Furthermore, transport regulations for crossing borders in the EU, which might occur if we export cultivation of our GMMs to another country, would mean we must adhere to EU GMO border rules in <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>, which implements the Cartagena Protocol.<br />
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<p><br />
At Reading, we would also have to submit an application to at least 3 committees before getting our proposal approved, and speak to the building manager for each building we would want to get our panel put on. Although the committees sound like more regulatory hurdles, the university safety officers would contact HSE for us, and we would supply all our information to the safety officers, making the process much easier. Furthermore, the biological safety committee would be the committee we would contact for the expert advice required when carrying out a risk assessment, and all the buildings at the university count as one private property, for which only one application to HSE needs to be made.<br />
</p><br />
<p><br />
Commercialisation of our product would mean selling it as a new source of renewable energy. Renewable fuel sources are subject to other rules in the EU, which stipulate criteria that must be met for a source to be labelled as “renewable”. The Fuel Quality Directive (FQD) is relevant to renewable fuels for transport, such as biofuels, and the Renewable Energy Directive (RED) is pertinent to other energy sources that wish to be labelled as renewable; ours could fall under the latter. The requirements in RED are essentially requirements for EU member states, but in order for the standards to be met, it is individual companies that ultimately must comply. The RED is enforced by the European Commission Directorate General for Energy. The requirements include showing a reduction in greenhouse gas emissions over the course of the fuel’s production, and using life cycle analysis (LCA) methods to calculate the “carbon intensity” of our energy source. Our “sustainability analysis” must encompass other features beyond an LCA. To meet the specifications in the RED, we must check 12 independent factors of our energy source (including its production and transportation), and have this verified by a third party. The analysis method must be approved by the EU.<br />
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For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.<br />
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<p><br />
Meeting experts in GMO safety and regulations, and consulting the appropriate legislation, has brought a number of key findings to light. Perhaps the most interesting is that current rules are not set up to cover a project like ours, that involves a container of GMMs outside and building, possibly on a person’s private property. As the Scientific Committees that in the EC are <a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">currently reviewing the risks of synthetic biology</a>, and that GMO-devices may become more common in the future, we may see regulations adapting more to cover these areas in the coming years.<br />
</p><br />
<p><br />
Although there are clearly areas where our panel counts as non-standard use, and so might be reclassified as higher risk than class 1, passing regulations may not be our biggest hurdle in getting our technology to the market. Our system complies with the safety measures needed, and our organism is of no or negligible risk to human safety or health, or the environment. Other obstacles that might be difficult to pass include how heavy our panels will be, and whether this will be a problem for transport or installing on rooftops, as having employees or members of the public on roofs would be a large safety risk in itself. The cost of submitting contained use applications to HSE also needs to be taken into account, and how the panels will be maintained without trained personnel on site. Furthermore, aspects of scaling up our technology also need to be worked out, such as what the optimal ratio of cyanobacteria-inoculated media to potassium ferricyanide is, and whether our cyanobacteria will survive long-term use in a photovoltaic cell. In addition, we do not know if people will be interested in continuing to pay for new media that must be added. By contrast, normal photovoltaic cells are one-off payments, even if they would be much more expensive than a cyanobacterial photovoltaic cell.<br />
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<p><br />
This is a bullet-point guide of what we’d need to do, step-by-step, to get our product to the market. It also provides a summary of our findings. It is by no means exhaustive though. We have focussed on the panels, rather than other aspects of our hypothetical business, and expect that many hurdles would magically appear to make life more difficult if we attempted to carry out our proposal.<br />
</p><br />
<p><br />
<ul><br />
<li>Get proof that our organisms our less fit (competition assay)</li><br />
<li>Get proof that our system is secure (leaks)</li><br />
<li>Carry out a risk assessment</li><br />
<li>Get advice from an expert person or panel on the risk assessment</li><br />
<li>Provide appropriate containment measures</li><br />
<li>Review the class of our work in respect to the containment measures</li><br />
<li>Draw up an emergency plan, inform the relevant personnel.</li><br />
<li>Submit an application to HSE, pay £452 for each site</li><br />
<li>Wait 10 days for acknowledgement of receipt</li><br />
<li>Commence work</li><br />
</ul><br />
<p><br />
Before any of this, it would be advisable to contact HSE to for advice on our proposition, however.<br />
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<p><br />
In this report we mainly referred to a few pieces of legislation, conversations we had with experts, and other sections of our wiki. As such, it made a lot more sense to link to all our resources as we went along, rather than using a Harvard or Vancouver style of referencing. However, we realise that it’s also convenient to have all the resources or references in one section. Here is a list of resources we used. If you’re hoping to review regulations on GMMs in the EU, this should be your starting point. <br />
</p><br />
<br /><br />
<p class="title"><i>Worldwide</i></p><br />
<p><br />
<b>The Cartagena Protocol</b> - UN-ratifed agreement for transborder GMO movement<br />
</p><br />
<br /><br />
<p class="title"><i>EU Directives</i></p><br />
<p><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009L0041&from=EN">Directive 2009/41/EC</a></b> - contained use <br /><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0018&from=EN">Directive 2001/18/EC</a></b> - deliberate release <br /><br />
<b>Directive 2000/54/EC</b> - risk classification of organisms <br /><br />
<b>Regulation EC 1946/2003</b> - transborder movement of GMOs. Implements the Cartagena Protocol <br /><br />
<b><a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">Opinion on Synthetic Biology</a></b> - first in a series to start reviewing risk in synthetic biology <br /><br />
For other EU legislation, start here <br /><br />
</p><br />
<br /><br />
<p class="title"><i>UK Regulations</i></p><br />
<p><br />
<b><a href="http://www.hse.gov.uk/pubns/books/l29.htm">Contained Use</a></b> <br /><br />
<b><a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/">SACGM Compendium of Guidance</a></b> <br /><br />
<b>Other UK rules</b> are mentioned <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">here</a> <br /><br />
<br />
</p><br />
<br /><br />
<p class="title"><i>Other</i></p><br />
<p><br />
We also found the <a href="http://biofuelpolicywatch.wordpress.com">BioFuel Policy Watch</a> blog and <a href="http://dglassassociates.wordpress.com">its associated blog</a> to be useful for general information. David Glass’s blog post on <a href="http://dglassassociates.wordpress.com/2013/09/22/regulation-of-industrial-use-of-algae-or-cyanobacteria-in-europe-part-1/">EU regulations for algae and cyanobacteria</a> is also a great starting point.<br />
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<p><br />
When we first asked the question, “can we put our bacteria on a roof?”, we didn’t envision giving such a detailed response. The proposition only reached its current form through repeated rounds of meetings with the students and supervisors, time spent reading EU and UK regulations and, most importantly, meetings with experts in biosafety at Reading. Gretta Roberts and Professor Jim Dunwell were very kind in giving up their time to answer all our questions, and we are very grateful for their input.<br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/Human_PracticesTeam:Reading/Human Practices2014-10-17T22:10:55Z<p>Seafloor: </p>
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An important part of iGEM is thinking about the wider impact of your project. We considered whether it would be possible to set up our cyanobacterial solar panels on roofs at Reading or on people’s houses. This meant coming up with a <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">design for a larger photovoltaic cell</a>, considering the biosafety issues involved, and what regulatory challenges we would face.<br />
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<p><img id="methods" align=centre src="https://static.igem.org/mediawiki/2014/3/30/Cyano_cultures.jpg" width="800px"></p><br />
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<li><a href="#summary">Summary</a></li><br />
<li><a href="#intro">Introduction</a></li><br />
<li><a href="#levels">Levels of Regulation</a></li><br />
<li><a href="#eu">EU Regulations</a></li><br />
<ul><br />
<li><a href="#euintro">Introduction</a></li><br />
<li><a href="#eucontained">Contained Use</a></li><br />
<li><a href="#eudelib">Deliberate Release</a></li><br />
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<li><a href="#uk">UK Regulations</a></li><br />
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<li><a href="#ukintro">Introduction</a></li><br />
<li><a href="#ukcontained">Contained Use</a></li><br />
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<li><a href="#other">Other Regulations</a></li><br />
<li><a href="#conc">Findings and Conclusions</a></li><br />
<li><a href="#road">The Roadmap</a></li><br />
<li><a href="#resources">Resources</a></li><br />
<li><a href="#acknowledgements">Acknowledgements</a></li><br />
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<p><br />
Creating a cyanobacterial photovoltaic cell and getting it installed on a roof at a university presents a number of challenges. The design of the system is the first aspect to consider. We look into design and cost of the parts on the Fuel Cell page. Then there are European Union (EU) and government regulations and, in the case of Reading, several boards and internal committees through which applications would have to pass. We consider these in this section. Each of these raises questions about biosafety, such as potential effects of the escape of our organism into surrounding environments. In addition to using our technology at our own university, we also considered commercialising the technology and installing it on people’s houses. This opens up a new realm of issues, such as getting our energy source classed as renewable according to the EU’s Renewable Energy Directive (RED), and getting permission for having GMOs on many distinct properties. These wider problems are also reviewed here.<br />
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<p><br />
Many sections of our report will be applicable to other teams considering contained use of GMOs, and we hope future teams will benefit from our research. The EU section will be particularly relevant to other EU member states, as the EU regulations form the common minimum requirements for each country. In general, this page should guide teams considering biosafety issues associated with cyanobacteria; there are currently no reviews of biosafety in synthetic biology of cyanobacteria that we are aware of. We finish with a roadmap for those thinking about whether they could commercialise a genetically modified microorganism (GMM)-containing system, especially as a renewable fuel source.<br />
</p><br />
<p><br />
It should be noted that the report mainly refers to contained use of GMMs. Though our system is contained, parts of it could be considered to overlap with deliberate release. We have therefore focussed on regulations pertaining to contained use, but have referred to those on deliberate release where our system could potentially fall under its purview. Due to this relevance, and partly time due to time constraints, we have not exhaustively considered deliberate release or contained use categorised as class 2 or above.<br />
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<p><br />
At the highest level, the Cartagena Protocol on Biosafety covers living modified organisms (LMOs) and their transport across borders. This is an international United Nations agreement that has been in place since 2003, and is implemented in the EU by <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>. Below that, the EU issues “directives” on genetically modified organisms (GMOs) that must be implemented by all EU member states. For contained use, only the state’s regulations need to be considered; there is no involvement at the EU level. For deliberate release, rules are much more complicated, involving notification of the European Commision (EC), and will not be covered here. In the UK, the EU directives are implemented by the Department for Environment, Food and Rural Affairs (DEFRA) and the Health and Safety Executive (HSE). Finally, at Reading we would have to pass at least 3 committees - including the sub-committee for biological safety, the project committee, and the environmental committee - in addition to getting approval from the building manager.<br />
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<p class="title" id="euintro"><i>Introduction to EU Regulations</i></p><br />
<p><br />
The two EU directives concerning our plans are the directive 2009/41/EC on contained use and the 2001/18/EC directive on deliberate release of GMOs. Of these, the contained use directive is probably most appropriate. The 2009/41/EC directive defines contained use as: “any activity in which micro-organisms are genetically modified or in which such GMMs are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with, and to provide a high level of safety for, the general population and the environment”, in Article 2(c).<br />
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<p><br />
By contrast, the 2001/18/EC directive defines deliberate release as: “any intentional introduction into the environment of a GMO or a combination of GMOs for which no specific containment measures are used to limit their contact with and to provide a high level of safety for the general population and the environment”, in Article 2(3).<br />
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<p><br />
By comparing these, and reviewing <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the proposed implementation of our idea</a>, we can see that we would most likely fall under the contained use directive because of our suggested containment measures. Our technology will use all of the activities specified by contained use, and implement appropriate safety measures. Based on this, we shall chiefly address contained use regulations, but mention rules on environmental release that are significant.<br />
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<p class="title" id="eucontained"><i>EU: Contained Use</i></p><br />
<p><br />
In summary, class 1 contained use requires: <br /><br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment</li><br />
<li>classification of the risk of the GMM according to the assessment</li><br />
<li>appropriate containment</li><br />
<li>notification of the relevant authority</li><br />
<li>an emergency plan for accidental release</li><br />
</ul><br />
</p><br />
<p><br />
We shall explore each of these points in further detail.<br />
</p><br />
<p><br />
Article 4 defines one of the main requirements for contained use: for a risk assessment to be carried out (Article 4(2)), in accordance with the guidelines in Annex III, with the aim of classifying the GMM. The criteria include assessing the potential to cause disease, effect on the environment (Annex III (A1)), and harmful effects of the genetic material, recipient, donor, vector and final GMM (Annex III (A2)). The severity of these issues and the chance of them happening must also be analysed. As a non-pathogenic organism, capability of causing disease is not relevant to our organism. The most germaine section is in Annex III (A1), which lists considering “deleterious effects due to establishment or dissemination in the environment” and “deleterious effects due to the natural transfer of inserted genetic material to other organisms.” Annex III (B7) goes on to require that the proposed use of the microorganism be combined with the above assessment in assigning it to a class. “Non-standard operations” is mentioned as affecting classification (Annex III (B7 iii)); this term is ambiguous, but may encompass our suggestion of having GMMs on roofs of private properties. <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">Our drip-in/drip-out system</a>, with the filter needing autoclaving upon replacement, may also fall under non-standard use. This would need to be taken into consideration if attempting to use our technology commercially. From this, it is clear that our GMM belongs in class 1 (as defined in Article 4(3)), so requires level 1 containment measures, though any doubt raised from our “non-standard operations” might cause a more strict classification (Article 4(4)). For those wishing to classify their organism, Directive 2000/54/EC can be referred to, or classification systems of the specific country.<br />
</p><br />
<p><br />
The risk assessment has a particular focus on waste disposal (Article 4(5)), making our drip-out waste disposal system very important. As removing the filter from the panel might be a source of accidental release, careful planning of waste management should be high on our priority list. The final risk assessment must be given to the competent authority (Article 4(6)) - HSE in the case of the UK. Containment measures are defined in Annex IV.<br />
</p><br />
<p><br />
Article 6 poses a potential issue. It requires notifying authorities upon contained use at each new property. While this is reasonable for single use at the university, our idea of having installations on separate houses would mean giving the information listed in Annex V for each site, including the risk assessment, which individuals are responsible for supervising, and a description of the premises. This would mean that the risk assessment must be sufficiently comprehensive to envision all potential environments where an installation may be set up, and would mean extra administration work for our organisation. It may be that, in the future, EU directives would need to be altered in order to make GMM technologies like ours more easily available for public benefit. Deliberate release regulations already contain a separate section for commercial use, and contained use may be separated this way in the future too.<br />
</p><br />
<p><br />
For class 1 organisms, no further notification is needed before commencing with contained use (Article 7). For higher risk classes more information is needed; as our organism is only class 1 we will not consider this, but rules can be found in Articles 8 and 9.<br />
</p><br />
<p><br />
Further thought should be given to the minimum containment measures stipulated in Annex IV, and whether our system meets these conditions. There are different requirements given for different potential situations. Our proposition would most likely fall under “Containment and other protective measures for other activities” for the panel itself, but other containment procedures would need to be reviewed for labs where genetic modification is done and areas where GMMs are cultured. Almost all containment options for class 1 organisms in this category are optional according the Annex IV. It is likely that the level of containment assumed for this category is more severe than in our proposition (i.e. the EC has assumed all contained use will occur inside a building). As such, we should expect to see some or all of the containment measures to be required, rather than optional, for our project. <br />
</p><br />
<p><br />
Beyond the obvious physical containment, there are several possible containment measures we could be required to enforce. This includes control of aerosols during “addition of material to a closed system or transfer of material to another system”. This would include transferring cyanobacteria to the fuel cell, which would be done in a separate contained facility, and during the removal of waste or the waste filter <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">(see design section)</a>, which would have to be done on site. The latter is one of the biggest issues our project could face.<br />
</p><br />
<p><br />
Inactivation of waste containing GMMs is also listed as optional, but could potentially be required. Furthermore, the air leaving the system, assuming filter-sterilised air is bubbled through our panel, could have to be filtered to prevent or minimise release. The only point already required for class 1 work is that personnel wear protective clothing.<br />
</p><br />
<p><br />
According to Article 13, an emergency plan is required. This must be made available to the public, relevant bodies and authorities, and other concerned EU member states. The plan is required in case containment measures fail, leading to “serious danger, whether immediate or delayed, to humans outside the premises and/or to the environment”. No information is given on how extensive this plan should be, and no minimum requirements are given. Member state legislation must therefore be consulted for any rules on how the plan must be structured.<br />
</p><br />
<p><br />
Finally, it should be noted that member states may consult the public on the proposition if they decide it is relevant (Article 12).<br />
</p><br />
<br /><br />
<p class="title" id="eudelib"><i>EU: Deliberate Release</i></p><br />
<p><br />
Below is outlined some of the salient points from the EU directive on deliberate release. These may be useful to other teams reviewing regulations. The key points are:<br />
</p><br />
<p><br />
<ul><br />
<li>a risk assessment is required (Article 4; Annex III)</li><br />
<li>regulations are different for commercial and non-commercial use</li><br />
<li>for non-commercial GMO work (Part B):</li><br />
<ul><br />
<li>parties must give a risk assessment and monitor use and effects</li><br />
<li>the authority can tell the public</li><br />
<li>approved uses must be reported to the EU</li><br />
</ul><br />
<li>for commercial GMO work (Part C):</li><br />
<ul><br />
<li>parties must notify the relevant authority before placing the product on the market</li><br />
<li>putting it on the market is defined as making it available to 3rd parties</li><br />
<li>the authority produces an “assessment report”</li><br />
<li>this is given to the applicant, the EC and EU member states</li><br />
<li>decisions apply throughout the EU</li><br />
<li>the public must be notified</li><br />
<li>as of June 2014, <a href="http://ec.europa.eu/food/plant/gmo/legislation/future_rules_en.htm">member states can restrict or ban GMOs in their country</a> that have been approved for all states</li><br />
</ul><br />
</ul><br />
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<td colspan="3"><h3 class="title" id="uk">UK Regulations</h3><br />
<p class="title" id="ukintro"><i>Introduction to UK Regulations</i></p><br />
<p><br />
<p><br />
The EU regulations are useful to us as they provide a baseline level of regulation we can expect if we try to implement our technology anywhere in the EU. In each EU member state, it is ultimately that country’s regulations which we must abide by. We will now consider what the regulations are like the UK. <br />
</p><br />
<p><br />
In general, they are slightly stricter than the basic EU regulations. The main regulations are <a href="http://www.hse.gov.uk/pubns/priced/l29.pdf">the HSE Contained Use regulations</a>, which are newly updated for 2014, and the accompanying SACGM Compendium of Guidance, which has yet to be updated to meet the new Contained Use document. Along with this, there are <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the regulations on deliberate release</a> from 1997, <a href="http://www.legislation.gov.uk/ukpga/1990/43/contents">section 108(1) of the Environment Protection Act </a> from 1990, and <a href="http://www.legislation.gov.uk/uksi/1996/1106/contents/made">the Genetically Modified Organisms Regulations </a> (1996) that are related to GMOs. The latter three are all focussed on environmental release, so won’t be covered here. Regulations are upheld by the HSE and DEFRA. <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">The HSE website</a> can be consulted for all other regulations that might relate to the use of GMOs.<br />
</p><br />
<br /><br />
<p class="title" id="ukcontained"><i>UK Regulations: Contained Use</i></p><br />
<p><br />
The essential requirements for contained use in the UK are in the line with EU rules; we need to carry out a risk assessment and classify our organism, and notify the HSE before commencing GM work. So far we have only talked about the sites where the panels will be installed, but we will also have to consider regulations for handling, transport, work area decontamination, inactivation of GMMs and their disposal (including waste management). While the GMMs’ safety would need to be assessed by HSE, the system containing them, our panel, will need to be tested for leakages, with this evidence submitted to DEFRA.<br />
</p><br />
<p><br />
The definition given for contained use in Part 1, regulation 2 is “an activity in which organisms are genetically modified or in which genetically modified organisms are cultured, stored, transported, destroyed, disposed of or used in any other way and for which physical, chemical or biological barriers, or any combination of such barriers, are used to limit their contact with, and to provide a high level of protection for, humans and the environment”. Also in part 1, regulations 26 specifically tells us that commercial disposal of waste containing GMOs also falls under contained use. The contained use definition is similar to the EU definition, but is slightly more specific about what the containment measures must entail. In Part 1, paragraphs 24 and 45 give examples of what the barriers for our system might be expected to be. Physical could include a container, which would be the panel itself in our case. Chemical barriers may cover inactivation before waste disposal, and biological would include attenuating characteristics that debilitate the organism so that it is “rendered unable to survive outside of a specialised environment”. These barriers are discussed further in the safety page.<br />
</p><br />
<p><br />
The first requirement to consider is the risk assessment. For GMMs this is covered in Part 2, regulation 5, with more details on the assessment in Schedule 3 (Part 2). More emphasis is placed on risk to human and health and environment than in EU regulations. Regulation 5, paragraph 43, also answers questions we raised in the “EU: Contained Use” section about whether we could apply the same risk assessment to multiple sites with the same roof installation: “Where the contained use is identical at the multiple sites (eg in a clinical trial), the same risk assessment may apply to all the sites”. However it does point out that local changes in practices need to be taken into account. For us, practices would remain the same, but the surrounding environment may be different (e.g. there may be a pond at one property, where our organisms could theoretically survive). What the risk assessment must encompass is introduced in paragraph 44. In short, we must outline our plans, potential harmful effects, the chance of them occurring and their severity, and how we’ll deal with waste. These topics are covered in the safety page, where we look at each of our mutations; waste disposal is covered in <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">the Fuel Cell page</a>.<br />
</p><br />
<p><br />
It is clear that the detail needed in the risk assessment is partly defined by how well-understood the microorganism and mutations are. Although our organism is clearly a non-pathogenic, non-hazardous class 1 organism, it is not as well understood as Escherichia coli K-12, for example, and the genes are not ones that are commonly used. They do not have a strong history of safe use, like green fluorescent protein (GFP). All our mutations have been done before, however, while measuring for different endpoints, so literature is available for reference on the effects of our mutations. From paragraphs 52 and 53, we know that the classification of the work changes to reflect the level of containment needed. When considering EU regulations, we were unsure of the extent of containment required, as our organism is class 1, but is used in unusual and potentially problematic environments. Under UK regulations, it appears our work could be reclassified to class 2 if we deem the containment measures for class 2 work to be desirable. This brings in a previously unforeseen hurdle: our work may be relabelled as higher than class 1 because of the containment measures needed on private properties with no trained personnel. We will continue to assume our work is class 1, but mention class 2 rules where appropriate.<br />
</p><br />
<p><br />
In summary, for the risk assessment we must identify hazards, assign appropriate containment measures, then reclassify our work based on these (if necessary). These instructions are similar to, but more detailed than, those for EU member states in general.<br />
</p><br />
<p><br />
According to regulation 8, we need to obtain advice from a person or committee on the risk assessment. If our organism were reclassified as class 2, this would have to be a biological safety committee. At Reading this would not be an issue, as there is already a committee from which we could obtain advice. If the classification remained as class 1, our meeting with Gretta Roberts and Professor Jim Dunwell, who have advised us with regards to regulations and safety, should be sufficient.<br />
</p><br />
<p><br />
Regulation 9 requires notification of premises to be employed for contained use. We must first submit the information in Schedule 5, then wait 10 days for a response. A single notification may include more than 1 premises in situations where more than one premise is owned by the same company, so it’s possible that multiple sites could be asked about at once. This would reduce the cost of notifying HSE, which is currently <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£472 for class 1</a> and <a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmwarn.htm">£943 for class 2</a>. However, if our organism still falls under class 1, we only need to submit a summary of the risk assessment, details on waste management and the advice we received during the risk assessment, and confirmation that relevant authorities will be notified of the emergency plan. The rest of the information needed is basic details like the address of the premises. For class 2 work, regulation 10 should be consulted.<br />
</p><br />
<p><br />
Part 3 outlines practices that must occur for contained use. Regulations 18, 19, 21 and 22 are all relevant to different stages of our plans. Sections relevant specifically to the panels include paragraphs 107-109, which tell us that containment measures must be tested; this may involve checking each panel for defects or leaking before deployment, and possibly visiting sites at intervals to check they are still functioning correctly (paragraph 108). This could mean simply looking over the panel for any cracks or leakages to check for physical containment. Checking that biological containment is still in place, for example by checking that cells cannot directly transfer or uptake DNA after the pilT1 mutation, may involve taking a sample from panels. This itself means removing liquid containing GMMs and transporting it back to the lab for testing transformation efficiency; we would have to ensure that taking any liquid samples would not have any risks of accidental release. It is unlikely, but possible, that we could be required to check for our GMM in the surrounding environment to ensure there had been no release (paragraph 111).<br />
</p><br />
<p><br />
Regulation 19 specifically covers containment measures for GMMs. Containment measures must be reviewed “at regular intervals” (paragraphs 128 and 129), which must occur more frequently for non-standard work. According to paragraph 134, our organism does not fit the criteria for class 1 work that does not require waste inactivation, because our organism does not contain “multiple disabling mutations”, so waste from our panel must be inactivated. According to 135, it is acceptable to take the filter to another location and autoclave it, assuming steps are taken to make sure storage and transport are safe, and that the process is effective at inactivating our GMM.<br />
</p><br />
<p><br />
We must follow Schedule 8, Part 2, Table 2 for containment measures. As with our assessment of EU containment requirements, we have chosen the “other” section, as this seems most appropriate. Here the only absolute requirement is that personnel must wear work clothing. Measures that might be required, if deemed so by the risk assessment, include: physical separation, control of aerosols (as discussed in the EU contained use section), inactivation of waste or removed fluid, and control for spillage. The last of these is the only one not fully considered so far in our design.<br />
</p><br />
<p><br />
Emergency plans are discussed in regulation 21. According to paragraph 139, however, “an emergency plan should only be prepared for work with organisms that pose the highest hazards to humans or the environment.” Although our organism is low hazard, the risk is perhaps higher because the GMMs are not contained in a facility. As only hazard is mentioned, it is possible that we would not have to draw up an emergency plan (for our organism the hazard is low, but the risk slightly higher as it is not contained in a building). Furthermore, only those on the premises would be exposed to our non-dangerous GMM, but paragraph 139 only requires an emergency plan when the health or safety of those outside the premises is in danger. This probably assumes the work is a designated building with trained personnel on site, though. Given this, and our non-standard plans, it would be prudent to anticipate an emergency plan being required. If this were the case, it must be submitted along with our contained used application to HSE (paragraph 140). Paragraph 141 covers the requirement for an emergency plan; those “on the site affected by the plan” should know the plan, so residents in houses where a panel is installed, or building managers for a university building, would have to be familiar with the plan (paragraph 142). The plan must also be publicly available (paragraph 143), as we know from the EU requirements. Although we have primarily talked about the risk to the environment of our GMMs, we should also consider the risks of releasing potassium ferricyanide to the environment (a part of our solar panel), like how much would be released, any risks to human health or safety, and any risks to the environment.<br />
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<td colspan="3"><h3 class="title" id="other">Other Regulations</h3><br />
<p><br />
We’ve covered the main EU and UK regulations regarding the installation of our actual panels, but there are many other regulations relevant to our plans. Contained use regulations are the main ones, which would cover the genetic modification of our organisms in a lab, the transport of our organisms to a facility where they can be grown up, that facility itself, transport to houses or the university, and the property where the panel is installed. There are other regulations that could come into play, however. This includes rules on the <a href="http://www.hse.gov.uk/cdg/introduction.htm">carriage of dangerous goods</a>, which might include our GMMs or potassium ferricyanide. Furthermore, transport regulations for crossing borders in the EU, which might occur if we export cultivation of our GMMs to another country, would mean we must adhere to EU GMO border rules in <a href="http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003R1946:EN:NOT">Regulation EC 1946/2003</a>, which implements the Cartagena Protocol.<br />
</p><br />
<p><br />
At Reading, we would also have to submit an application to at least 3 committees before getting our proposal approved, and speak to the building manager for each building we would want to get our panel put on. Although the committees sound like more regulatory hurdles, the university safety officers would contact HSE for us, and we would supply all our information to the safety officers, making the process much easier. Furthermore, the biological safety committee would be the committee we would contact for the expert advice required when carrying out a risk assessment, and all the buildings at the university count as one private property, for which only one application to HSE needs to be made.<br />
</p><br />
<p><br />
Commercialisation of our product would mean selling it as a new source of renewable energy. Renewable fuel sources are subject to other rules in the EU, which stipulate criteria that must be met for a source to be labelled as “renewable”. The Fuel Quality Directive (FQD) is relevant to renewable fuels for transport, such as biofuels, and the Renewable Energy Directive (RED) is pertinent to other energy sources that wish to be labelled as renewable; ours could fall under the latter. The requirements in RED are essentially requirements for EU member states, but in order for the standards to be met, it is individual companies that ultimately must comply. The RED is enforced by the European Commission Directorate General for Energy. The requirements include showing a reduction in greenhouse gas emissions over the course of the fuel’s production, and using life cycle analysis (LCA) methods to calculate the “carbon intensity” of our energy source. Our “sustainability analysis” must encompass other features beyond an LCA. To meet the specifications in the RED, we must check 12 independent factors of our energy source (including its production and transportation), and have this verified by a third party. The analysis method must be approved by the EU.<br />
</p><br />
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<td colspan="3"><h3 class="title" id="conc">Findings and Conclusions</h3><br />
<p><br />
Meeting experts in GMO safety and regulations, and consulting the appropriate legislation, has brought a number of key findings to light. Perhaps the most interesting is that current rules are not set up to cover a project like ours, that involves a container of GMMs outside and building, possibly on a person’s private property. As the Scientific Committees that in the EC are <a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">currently reviewing the risks of synthetic biology</a>, and that GMO-devices may become more common in the future, we may see regulations adapting more to cover these areas in the coming years.<br />
</p><br />
<p><br />
Although there are clearly areas where our panel counts as non-standard use, and so might be reclassified as higher risk than class 1, passing regulations may not be our biggest hurdle in getting our technology to the market. Our system complies with the safety measures needed, and our organism is of no or negligible risk to human safety or health, or the environment. Other obstacles that might be difficult to pass include how heavy our panels will be, and whether this will be a problem for transport or installing on rooftops, as having employees or members of the public on roofs would be a large safety risk in itself. The cost of submitting contained use applications to HSE also needs to be taken into account, and how the panels will be maintained without trained personnel on site. Furthermore, aspects of scaling up our technology also need to be worked out, such as what the optimal ratio of cyanobacteria-inoculated media to potassium ferricyanide is, and whether our cyanobacteria will survive long-term use in a photovoltaic cell. In addition, we do not know if people will be interested in continuing to pay for new media that must be added. By contrast, normal photovoltaic cells are one-off payments, even if they would be much more expensive than a cyanobacterial photovoltaic cell.<br />
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<td colspan="3"><h3 class="title" id="road">The Roadmap</h3><br />
<p><br />
This is a bullet-point guide of what we’d need to do, step-by-step, to get our product to the market. It also provides a summary of our findings. It is by no means exhaustive though. We have focussed on the panels, rather than other aspects of our hypothetical business, and expect that many hurdles would magically appear to make life more difficult if we attempted to carry out our proposal.<br />
</p><br />
<p><br />
<ul><br />
<li>Get proof that our organisms our less fit (competition assay)</li><br />
<li>Get proof that our system is secure (leaks)</li><br />
<li>Carry out a risk assessment</li><br />
<li>Get advice from an expert person or panel on the risk assessment</li><br />
<li>Provide appropriate containment measures</li><br />
<li>Review the class of our work in respect to the containment measures</li><br />
<li>Draw up an emergency plan, inform the relevant personnel.</li><br />
<li>Submit an application to HSE, pay £452 for each site</li><br />
<li>Wait 10 days for acknowledgement of receipt</li><br />
<li>Commence work</li><br />
</ul><br />
<p><br />
Before any of this, it would be advisable to contact HSE to for advice on our proposition, however.<br />
</p><br />
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<td colspan="3"><h3 class="title" id="resources">Resources</h3><br />
<p><br />
In this report we mainly referred to a few pieces of legislation, conversations we had with experts, and other sections of our wiki. As such, it made a lot more sense to link to all our resources as we went along, rather than using a Harvard or Vancouver style of referencing. However, we realise that it’s also convenient to have all the resources or references in one section. Here is a list of resources we used. If you’re hoping to review regulations on GMMs in the EU, this should be your starting point. <br />
</p><br />
<br /><br />
<p class="title"><i>Worldwide</i></p><br />
<p><br />
<b>The Cartagena Protocol</b> - UN-ratifed agreement for transborder GMO movement<br />
</p><br />
<br /><br />
<p class="title"><i>EU Directives</i></p><br />
<p><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009L0041&from=EN">Directive 2009/41/EC</a></b> - contained use <br /><br />
<b><a href="http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0018&from=EN">Directive 2001/18/EC</a></b> - deliberate release <br /><br />
<b>Directive 2000/54/EC</b> - risk classification of organisms <br /><br />
<b>Regulation EC 1946/2003</b> - transborder movement of GMOs. Implements the Cartagena Protocol <br /><br />
<b><a href="http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_044.pdf">Opinion on Synthetic Biology</a></b> - first in a series to start reviewing risk in synthetic biology <br /><br />
For other EU legislation, start here <br /><br />
</p><br />
<br /><br />
<p class="title"><i>UK Regulations</i></p><br />
<p><br />
<b><a href="http://www.hse.gov.uk/pubns/books/l29.htm">Contained Use</a></b> <br /><br />
<b><a href="http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp/">SACGM Compendium of Guidance</a></b> <br /><br />
<b>Other UK rules</b> are mentioned <a href="http://www.hse.gov.uk/biosafety/gmo/law.htm">here</a> <br /><br />
<br />
</p><br />
<br /><br />
<p class="title"><i>Other</i></p><br />
<p><br />
We also found the <a href="http://biofuelpolicywatch.wordpress.com">BioFuel Policy Watch</a> blog and <a href="http://dglassassociates.wordpress.com">its associated blog</a> to be useful for general information. David Glass’s blog post on <a href="http://dglassassociates.wordpress.com/2013/09/22/regulation-of-industrial-use-of-algae-or-cyanobacteria-in-europe-part-1/">EU regulations for algae and cyanobacteria</a> is also a great starting point.<br />
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<td colspan="3"><h3 class="title" id="acknowledgements">Acknowledgements</h3><br />
<p><br />
When we first asked the question, “can we put our bacteria on a roof?”, we didn’t envision giving such a detailed response. The proposition only reached its current form through repeated rounds of meetings with the students and supervisors, time spent reading EU and UK regulations and, most importantly, meetings with experts in biosafety at Reading. Gretta Roberts and Professor Jim Dunwell were very kind in giving up their time to answer all our questions, and we are very grateful for their input.<br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:50:53Z<p>Seafloor: </p>
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<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</a>. For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.</p><br />
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<p>As part of our project we looked into whether it was possible to actually install a GM-cyanobacterial solar panel onto a roof. From this, we have learnt that information on the safety of our organism and our panel has to be sent to the Health and Safety Executive (HSE) and the Department for Environmental, Food and Rural Affairs (DEFRA). Information on our organism concerned carrying out a risk assessment in line with regulation 5 and Schedule 3, Part 2 of the HSE Contained Use regulations for the UK.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:50:42Z<p>Seafloor: </p>
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<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</a>.</p><br />
<p>For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.</p><br />
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<p>As part of our project we looked into whether it was possible to actually install a GM-cyanobacterial solar panel onto a roof. From this, we have learnt that information on the safety of our organism and our panel has to be sent to the Health and Safety Executive (HSE) and the Department for Environmental, Food and Rural Affairs (DEFRA). Information on our organism concerned carrying out a risk assessment in line with regulation 5 and Schedule 3, Part 2 of the HSE Contained Use regulations for the UK.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:49:02Z<p>Seafloor: </p>
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<td width="80%" valign="top"> <br />
<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</a>.</p><br />
<br /><br />
<p>For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.</p><br />
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<p>As part of our project we looked into whether it was possible to actually install a GM-cyanobacterial solar panel onto a roof. From this, we have learnt that information on the safety of our organism and our panel has to be sent to the Health and Safety Executive (HSE) and the Department for Environmental, Food and Rural Affairs (DEFRA). Information on our organism concerned carrying out a risk assessment in line with regulation 5 and Schedule 3, Part 2 of the HSE Contained Use regulations for the UK.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:43:12Z<p>Seafloor: </p>
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<tr><br />
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<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</a>.</p><br />
<br /><br />
<p>For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:42:22Z<p>Seafloor: </p>
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<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</a>.</p><br />
<br /><br />
<p>For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:40:03Z<p>Seafloor: </p>
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<td width="80%" valign="top"> <br />
<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</a>.</p><br />
<br /><br />
<p>For the final word on safety, check out <a href="https://igem.org/Team.cgi?id=1476">our official team page</a> and look through the safety section.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:35:40Z<p>Seafloor: </p>
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<p>Welcome to the safety page. Here we'll talk about the biosafety of our organism, and some of the issues associated with our <a href="https://2014.igem.org/Team:Reading/Fuel_Cell#panel">rooftop panel</p><br />
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<p>Let's get started.</p><br />
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{{Tail}}</div>Seafloorhttp://2014.igem.org/Team:Reading/SafetyTeam:Reading/Safety2014-10-17T21:33:02Z<p>Seafloor: </p>
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<p>Try not to get too excited. Here you'll find all things safety: links to safety forms, discussions about environmental release, the whole shebang. Yup, this is really happening. I know, just breathe. We'll talk you through it.</p><br />
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{{Tail}}</div>Seafloor