Team:Melbourne/Safety

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<a href="https://2014.igem.org/Team:Melbourne"style="color:#000000">Home </a> </td>
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Home</a> </td>
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Team </a> </td>
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Team</a> </td>
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<a href="https://igem.org/Team.cgi?year=2014&team_name=Melbourne"style="color:#000000">Official Team Profile </a></td>
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<a href="https://2014.igem.org/Team:Melbourne/Project"style="color:#000000">
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Project</a></td>
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Project</a> </td>
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<a href="https://2014.igem.org/Team:Melbourne/Parts"style="color:#000000">
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<a href="https://2014.igem.org/Team:Melbourne/Human_Practices"style="color:#000000">  
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Parts</a></td>
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Human Practices</a></td>
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<a href="https://2014.igem.org/Team:Melbourne/Modeling"style="color:#000000">
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<a href="https://2014.igem.org/Team:Melbourne/Achievements"style="color:#000000">
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Modeling</a></td>
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Achievements</a></td>
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Notebook</a></td>
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Notebook</a></td>
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<a href="https://2014.igem.org/Team:Melbourne/Protocols"style=" color:#000000">
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Protocols</a></td>
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<a href="https://2014.igem.org/Team:Melbourne/Safety"style="color:#000000">
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Safety</a></td>
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<a href="https://2014.igem.org/Team:Melbourne/Safety"style=" color:#000000">
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<a href="https://2014.igem.org/Team:Melbourne/Sponsors"style="color:#000000">
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Safety </a></td>
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Sponsors</a></td>
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<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000">
<a href="https://2014.igem.org/Team:Melbourne/Attributions"style="color:#000000">
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Attributions </a></td>
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Attributions</a></td>
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<td align="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td>
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<td align="right"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="46px"></a> </td>
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<h1 >University of Melbourne<br>
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<h1 >Safety</h1>
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iGEM team </h1>
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<ul>
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<ol>
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   <li><h3>1. Safety  Level Rating System</h3></li>
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   <li><strong>Safety  Level Rating System</strong></li>
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<p> Australia uses a four-part &lsquo;Safety  Level&rsquo; rating system for laboratories in which Level 4 is used for the most  dangerous organisms. Our laboratory is Level 2 (moderate risk).</p>
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<p>Australia uses a four-part &lsquo;Safety  Level&rsquo; rating system for laboratories in which Level 4 is used for the most  dangerous organisms. Our laboratory is Level 2 (moderate risk).</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
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   <li><strong>Work  Environments Used to Handle Biological Materials</strong></li>
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   <li><h3>2. Work  Environments Used to Handle Biological Materials</h3></li>
<p>Open benches and a laminar flow  hood/biosafety cabinet with open front are used when handling biological  materials in our laboratory.</p>
<p>Open benches and a laminar flow  hood/biosafety cabinet with open front are used when handling biological  materials in our laboratory.</p>
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<p>&nbsp;</p>
<p>&nbsp;</p>
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   <li><strong>Personal  Protective Equipment</strong></li>
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   <li><h3>3. Personal  Protective Equipment</h3></li>
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<p>In our laboratory lab coats, gloves and safety glasses/goggles are worn at all times and full face shields are worn  when using the UV light box. Latex gloves are used for most procedures in the  lab, however, nitrile gloves are worn when handling EtBr.</p>
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<p>In our laboratory lab coats, gloves and safety glasses/goggles are worn at all times and full face shields are worn  when using the UV light box. Latex gloves are used for most procedures in the  lab, however, nitrile gloves are worn when handling EtBr.</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
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   <li><strong>Disposal  of Biological Waste</strong></li>
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   <li><h3>4. Disposal  of Biological Waste</h3></li>
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<p>Biological waste (e.g. pipette tips)  are disposed of in a yellow biohazard which is sealed when not in use. When  full, this biohazard bin is sent to be autoclaved before it is disposed with  appropriately along with the biological waste from the other laboratories in  the institute.</p>
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<p>Biological waste (e.g. pipette tips)  is disposed of in a yellow biohazard which is sealed when not in use. When  full, this biohazard bin is sent to be autoclaved before it is disposed of appropriately along with the biological waste from the other laboratories in  the institute.</p>
<p>Biological waste such as liquid cell  cultures has bleach added to it and sits overnight in the fume cabinet before  the liquid is poured down a special biological waste drain and the container is  autoclaved.</p>
<p>Biological waste such as liquid cell  cultures has bleach added to it and sits overnight in the fume cabinet before  the liquid is poured down a special biological waste drain and the container is  autoclaved.</p>
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<p>&nbsp;</p>
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   <li><strong>Safety  Training</strong></li>
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   <li><h3>5. Safety  Training</h3></li>
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<p><strong>&nbsp;</strong></p>
 
<p>The Melbourne iGEM team received  safety training provided by The Bio21 Institute. The topics covered in this  training included: details of emergency actions, the university OH&amp;S policy  and issue resolution procedures, the university's environment policy and  procedures, procedures for reporting incidents/near misses, hazards and unsafe  acts/work processes, introduction to Bio21 EHS staff, location of relevant EHS  documentation, security, 'out of normal hours' access, equipment operating  procedures, general safety.</p>
<p>The Melbourne iGEM team received  safety training provided by The Bio21 Institute. The topics covered in this  training included: details of emergency actions, the university OH&amp;S policy  and issue resolution procedures, the university's environment policy and  procedures, procedures for reporting incidents/near misses, hazards and unsafe  acts/work processes, introduction to Bio21 EHS staff, location of relevant EHS  documentation, security, 'out of normal hours' access, equipment operating  procedures, general safety.</p>
<p>The laboratory safety requirements of  the institution can be found here: <a href="https://intranet.mdhs.unimelb.edu.au/ohse-training-requirements">https://intranet.mdhs.unimelb.edu.au/ohse-training-requirements</a>.<br>
<p>The laboratory safety requirements of  the institution can be found here: <a href="https://intranet.mdhs.unimelb.edu.au/ohse-training-requirements">https://intranet.mdhs.unimelb.edu.au/ohse-training-requirements</a>.<br>
</p>
</p>
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<p>&nbsp;</p>
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   <li><strong>Local  Rules and Regulations</strong></li>
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   <li><h3>6. Local  Rules and Regulations</h3></li>
<p>Christian Rantzau (Bio21 Health &amp;  Safety Representative) is responsible for biological safety at our institution.  We briefly discussed the program with him and no concerns were raised, nor  changes made to the project. He ensured that we were appropriately trained in the  safety protocols and procedures of the institution.</p>
<p>Christian Rantzau (Bio21 Health &amp;  Safety Representative) is responsible for biological safety at our institution.  We briefly discussed the program with him and no concerns were raised, nor  changes made to the project. He ensured that we were appropriately trained in the  safety protocols and procedures of the institution.</p>
<p>The biosafety guidelines of The Bio21  Institute can be found at: <a href="http://www.bio21.unimelb.edu.au/bio-21-institute-intranet/environmental-health-and-safety/safety">http://www.bio21.unimelb.edu.au/bio-21-institute-intranet/environmental-health-and-safety/safety</a>.</p>
<p>The biosafety guidelines of The Bio21  Institute can be found at: <a href="http://www.bio21.unimelb.edu.au/bio-21-institute-intranet/environmental-health-and-safety/safety">http://www.bio21.unimelb.edu.au/bio-21-institute-intranet/environmental-health-and-safety/safety</a>.</p>
<p>The regulations that govern biosafety  in research laboratories in Australia may be found via the following link: <a href="http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/legislation-2">http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/legislation-2</a>.</p>
<p>The regulations that govern biosafety  in research laboratories in Australia may be found via the following link: <a href="http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/legislation-2">http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/legislation-2</a>.</p>
 +
<p>&nbsp;</p>
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   <li><strong>Organisms  and Parts Used</strong></li>
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   <li><h3>7. Organisms  and Parts Used</h3></li>
<p>&nbsp;</p>
<p>&nbsp;</p>
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   <tr>
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     <td width="21%" nowrap valign="bottom"><p><strong>Species Name</strong></p></td>
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     <td width="168"><p><strong>Species Name</strong></p></td>
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     <td width="4%" nowrap valign="bottom"><p><strong>Risk Group</strong></p></td>
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     <td width="42"><p><strong>Risk<br>Group</strong></p></td>
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     <td width="6%" nowrap valign="bottom"><p><strong>Risk Group Source</strong></p></td>
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     <td width="78"><p><strong>Risk Group<br>Source</strong></p></td>
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     <td width="10%" nowrap valign="bottom"><p><strong>Disease risk to humans?</strong></p></td>
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     <td width="116"><p><strong>Disease risk<br>to humans?</strong></p></td>
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     <td width="15%" nowrap valign="bottom"><p><strong>Part number/name</strong></p></td>
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     <td width="213"><p><strong>How did you acquire it?</strong></p></td>
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    <td width="9%" nowrap valign="bottom"><p><strong>Natural function of part</strong></p></td>
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     <td width="151"><p><strong>How will you use it?</strong></p></td>
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    <td width="14%" nowrap valign="bottom"><p><strong>How did you acquire it?</strong></p></td>
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     <td width="17%" nowrap valign="bottom"><p>How will you use it?</p></td>
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   </tr>
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   <tr>
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     <td width="21%" nowrap valign="bottom"><p>E. coli, BL21(DE3)</p></td>
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     <td><p>E. coli, BL21(DE3)</p></td>
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     <td width="4%" nowrap valign="bottom"><p align="right">1</p></td>
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     <td><p align="right">1</p></td>
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     <td width="6%" nowrap valign="bottom"><p>E. coli K12</p></td>
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     <td><p>E. coli K12</p></td>
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     <td width="10%" nowrap valign="bottom"><p>Very low virulence</p></td>
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     <td><p>Very low virulence</p></td>
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     <td width="15%" nowrap valign="bottom"></td>
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     <td><p>Donation from neighboring lab</p></td>
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    <td width="9%" nowrap valign="bottom"></td>
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     <td><p>This is one of our chassis</p></td>
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    <td width="14%" nowrap valign="bottom"><p>Donation from neighboring lab</p></td>
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     <td width="17%" nowrap valign="bottom"><p>This is one of our chassis</p></td>
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   </tr>
   </tr>
   <tr>
   <tr>
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     <td width="21%" nowrap valign="bottom"><p>E. coli, SHuffle T7 commercially    available cells</p></td>
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     <td><p>E. coli, SHuffle T7<br>commercially    available cells</p></td>
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     <td width="4%" nowrap valign="bottom"><p align="right">1</p></td>
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     <td><p align="right">1</p></td>
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     <td width="6%" nowrap valign="bottom"><p>E. coli B</p></td>
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     <td><p>E. coli B</p></td>
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     <td width="10%" nowrap valign="bottom"><p>Very low virulence</p></td>
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     <td><p>Very low virulence</p></td>
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     <td width="15%" nowrap valign="bottom"></td>
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     <td><p>Acquired from local New England <br>Biolabs    agent</p></td>
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    <td width="9%" nowrap valign="bottom"></td>
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     <td><p>This is one of our chassis</p></td>
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    <td width="14%" nowrap valign="bottom"><p>Acquired from local New England Biolabs    agent</p></td>
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     <td width="17%" nowrap valign="bottom"><p>This is one of our chassis</p></td>
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   </tr>
   </tr>
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</table>
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<br>
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<table width="601" border="1" cellpadding="0" cellspacing="0">
   <tr>
   <tr>
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     <td width="21%" nowrap valign="bottom"></td>
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     <td width="190"><p><strong>Part number/name</strong></p></td>
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    <td width="4%" nowrap valign="bottom"></td>
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     <td width="109"><p><strong>Natural function<br>of part</strong></p></td>
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    <td width="6%" nowrap valign="bottom"></td>
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     <td width="294"><strong>How did you acquire it?</strong></td>
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    <td width="10%" nowrap valign="bottom"></td>
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     <td width="15%" nowrap valign="bottom"><p>Synthetic peptide 1 based on a de novo    amino acid sequence</p></td>
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-
    <td width="9%" nowrap valign="bottom"><p>Not natural</p></td>
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     <td width="31%" nowrap colspan="2" valign="bottom"><p>Designed DNA from scratch; DNA    synthesised by Life Technologies</p></td>
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   </tr>
   </tr>
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   <tr>
   <tr>
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     <td width="21%" nowrap valign="bottom"></td>
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     <td><p>Magainin 1 Star Peptide</p></td>
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    <td width="4%" nowrap valign="bottom"></td>
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     <td><p>Not natural</p></td>
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    <td width="6%" nowrap valign="bottom"></td>
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     <td>DNA synthesised by Life Technologies</td>
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    <td width="10%" nowrap valign="bottom"></td>
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    <td width="15%" nowrap valign="bottom"><p>Synthetic peptide 2 based on a de novo    amino acid sequence</p></td>
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-
     <td width="9%" nowrap valign="bottom"><p>Not natural</p></td>
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-
     <td width="31%" nowrap colspan="2" valign="bottom"><p>Designed DNA from scratch; DNA    synthesised by Life Technologies</p></td>
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   </tr>
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   <tr>
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     <td width="21%" nowrap valign="bottom"></td>
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     <td><p>USP Peptide</p></td>
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    <td width="4%" nowrap valign="bottom"></td>
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     <td><p>Not natural</p></td>
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    <td width="6%" nowrap valign="bottom"></td>
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     <td>DNA synthesised by GenScript</td>
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    <td width="10%" nowrap valign="bottom"></td>
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    <td width="15%" nowrap valign="bottom"><p>Synthetic peptide 3 based on a de novo    amino acid sequence</p></td>
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-
     <td width="9%" nowrap valign="bottom"><p>Not natural</p></td>
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     <td width="31%" nowrap colspan="2" valign="bottom"><p>Designed DNA from scratch; DNA    synthesised by Genscript</p></td>
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   </tr>
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   <tr>
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     <td width="21%" nowrap valign="bottom"></td>
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     <td><p>Linear Magainin 1</p></td>
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    <td width="4%" nowrap valign="bottom"></td>
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     <td><p>Antimicrobial peptide</p></td>
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    <td width="6%" nowrap valign="bottom"></td>
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     <td>DNA synthesised by Genscript</td>
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    <td width="10%" nowrap valign="bottom"></td>
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    <td width="15%" nowrap valign="bottom"><p>Synthetic peptide 4 based on a de novo    amino acid sequence</p></td>
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-
     <td width="9%" nowrap valign="bottom"><p>Not natural</p></td>
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-
    <td width="31%" nowrap colspan="2" valign="bottom"><p>Designed DNA from scratch; DNA    synthesised by Genscript</p></td>
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-
  </tr>
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  <tr>
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-
    <td width="21%" nowrap valign="bottom"></td>
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-
    <td width="4%" nowrap valign="bottom"></td>
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-
    <td width="6%" nowrap valign="bottom"></td>
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-
    <td width="10%" nowrap valign="bottom"></td>
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-
    <td width="15%" nowrap valign="bottom"><p>Synthetic peptide 5 based on a de novo    amino acid sequence</p></td>
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-
     <td width="9%" nowrap valign="bottom"><p>Not natural</p></td>
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-
    <td width="31%" nowrap colspan="2" valign="bottom"><p>Designed DNA from scratch; DNA    synthesised by Genscript</p></td>
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  </tr>
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  <tr>
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-
    <td width="21%" nowrap valign="bottom"></td>
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-
    <td width="4%" nowrap valign="bottom"></td>
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-
    <td width="6%" nowrap valign="bottom"></td>
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    <td width="10%" nowrap valign="bottom"></td>
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    <td width="15%" nowrap valign="bottom"><p>TEV protease gene</p></td>
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    <td width="9%" nowrap valign="bottom"><p>Tobacco etch virus protease</p></td>
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    <td width="31%" nowrap colspan="2" valign="bottom"><p>Donation from neighboring lab</p></td>
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   </tr>
   </tr>
 +
</table>
</table>
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<p><strong>Risks  of Our Project Now</strong></p>
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 +
<p>&nbsp;</p>
 +
<li><h3>8. Risks  of Our Project Now</h3></li>
<p><u>Risk to the Safety and Health of Team  Members/People Working in the Lab</u><br>
<p><u>Risk to the Safety and Health of Team  Members/People Working in the Lab</u><br>
-
   <em>E. coli</em> has a low, but not  non-existent virulence. For this reason, we wear gloves and lab coats and wash  our hands when entering and leaving the labs. When analysing DNA agarose gels both the EtBr solution and the UV light box pose a risk to the health of our team  members. In these specific cases we wear nitrile gloves when handling the gel  and a face shield when operating the light box.</p>
+
   <em>E. coli</em> has a low, but not  non-existent virulence. For this reason, we wear gloves and lab coats and wash  our hands when entering and leaving the labs. When analysing DNA agarose gels both the EtBr solution and the UV light box pose a risk to the health of our team  members. In these specific cases we wear nitrile gloves when handling the gel  and a face shield when operating the light box.</p>
<p><u>Risk to the Safety and Health of the  General Public</u><br>
<p><u>Risk to the Safety and Health of the  General Public</u><br>
   <em>E. coli</em> has a low, but not  non-existent virulence. For this reason, we wear gloves and lab coats and wash  our hands when entering and leaving the labs. This helps to reduce the risk to  the safety and health of the general public.</p>
   <em>E. coli</em> has a low, but not  non-existent virulence. For this reason, we wear gloves and lab coats and wash  our hands when entering and leaving the labs. This helps to reduce the risk to  the safety and health of the general public.</p>
<p><u>Risk to the Environment</u><br>
<p><u>Risk to the Environment</u><br>
-
   When disposing of things such as  liquid cell culture, we sit them overnight in the fume cabinet with some bleach in the,. In the morning we then pour the liquid down the shoot in the fume  cabinet and autoclave the containers. We do not pour hazardous chemicals down the sink. All of these action minimise the risks to the environment.</p>
+
   When disposing of things such as  liquid cell culture, we sit them overnight in the fume cabinet with some bleach in them. We pour the liquid down the shoot in the fume  cabinet on the next day and autoclave the containers. We do not pour hazardous chemicals down the sink. All of these action minimise the risks to the environment.</p>
<p><u>Risks to Security Through Malicious  Mis-Use</u><br>
<p><u>Risks to Security Through Malicious  Mis-Use</u><br>
   Our project does not pose much of a  risk in terms of malicious mis-use by others, but to try and prevent this we  have key card access into both the building and the labs so that they cannot be  accessed by the general public.</p>
   Our project does not pose much of a  risk in terms of malicious mis-use by others, but to try and prevent this we  have key card access into both the building and the labs so that they cannot be  accessed by the general public.</p>
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   <li><strong>Risks  of Our Project in the Future</strong>
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   <li><h3>9. Risks  of Our Project in the Future</h3>
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     <p>If our project is successful it will  allow people to use the molecules we make as a platform technology for making  disulphide bonded peptides. These peptides could potentially be used for  harmful purposes. We don&rsquo;t currently have any design features to reduce the  risk of someone using our project for harmful purposes. In the future if it  were deemed necessary we could potentially try to limit the types of amino residues or groups that could be added to the arms of our peptide by native  chemical ligation.</p>
+
     <p>If our project is successful it will  allow people to use the molecules we make as a platform technology for making  disulphide bonded peptides. These peptides could potentially be used for  harmful purposes. We don&rsquo;t currently have any design features to reduce the  risk of someone using our project for harmful purposes. In the future if it  were deemed necessary we could potentially try to limit the types of amino acid residues or groups that could be added to the arms of our peptide by native  chemical ligation.</p>
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Latest revision as of 02:05, 18 October 2014

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Home Team Project Human Practices Achievements Notebook Protocols Safety Sponsors Attributions

Safety

  • 1. Safety Level Rating System

  • Australia uses a four-part ‘Safety Level’ rating system for laboratories in which Level 4 is used for the most dangerous organisms. Our laboratory is Level 2 (moderate risk).

     

  • 2. Work Environments Used to Handle Biological Materials

  • Open benches and a laminar flow hood/biosafety cabinet with open front are used when handling biological materials in our laboratory.

    Some materials are handled in different work environments, for example DNA gels containing EtBr are only used in designated areas and some procedures involving bacteria that need to be sterile are performed near an open flame, while most other procedures are performed at open benches.

     

  • 3. Personal Protective Equipment

  • In our laboratory lab coats, gloves and safety glasses/goggles are worn at all times and full face shields are worn when using the UV light box. Latex gloves are used for most procedures in the lab, however, nitrile gloves are worn when handling EtBr.

     

  • 4. Disposal of Biological Waste

  • Biological waste (e.g. pipette tips) is disposed of in a yellow biohazard which is sealed when not in use. When full, this biohazard bin is sent to be autoclaved before it is disposed of appropriately along with the biological waste from the other laboratories in the institute.

    Biological waste such as liquid cell cultures has bleach added to it and sits overnight in the fume cabinet before the liquid is poured down a special biological waste drain and the container is autoclaved.

     

  • 5. Safety Training

  • The Melbourne iGEM team received safety training provided by The Bio21 Institute. The topics covered in this training included: details of emergency actions, the university OH&S policy and issue resolution procedures, the university's environment policy and procedures, procedures for reporting incidents/near misses, hazards and unsafe acts/work processes, introduction to Bio21 EHS staff, location of relevant EHS documentation, security, 'out of normal hours' access, equipment operating procedures, general safety.

    The laboratory safety requirements of the institution can be found here: https://intranet.mdhs.unimelb.edu.au/ohse-training-requirements.

     

  • 6. Local Rules and Regulations

  • Christian Rantzau (Bio21 Health & Safety Representative) is responsible for biological safety at our institution. We briefly discussed the program with him and no concerns were raised, nor changes made to the project. He ensured that we were appropriately trained in the safety protocols and procedures of the institution.

    The biosafety guidelines of The Bio21 Institute can be found at: http://www.bio21.unimelb.edu.au/bio-21-institute-intranet/environmental-health-and-safety/safety.

    The regulations that govern biosafety in research laboratories in Australia may be found via the following link: http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/legislation-2.

     

  • 7. Organisms and Parts Used

  •  

    Species Name

    Risk
    Group

    Risk Group
    Source

    Disease risk
    to humans?

    How did you acquire it?

    How will you use it?

    E. coli, BL21(DE3)

    1

    E. coli K12

    Very low virulence

    Donation from neighboring lab

    This is one of our chassis

    E. coli, SHuffle T7
    commercially available cells

    1

    E. coli B

    Very low virulence

    Acquired from local New England
    Biolabs agent

    This is one of our chassis


    Part number/name

    Natural function
    of part

    How did you acquire it?

    Magainin 1 Star Peptide

    Not natural

    DNA synthesised by Life Technologies

    USP Peptide

    Not natural

    DNA synthesised by GenScript

    Linear Magainin 1

    Antimicrobial peptide

    DNA synthesised by Genscript

     

  • 8. Risks of Our Project Now

  • Risk to the Safety and Health of Team Members/People Working in the Lab
    E. coli has a low, but not non-existent virulence. For this reason, we wear gloves and lab coats and wash our hands when entering and leaving the labs. When analysing DNA agarose gels both the EtBr solution and the UV light box pose a risk to the health of our team members. In these specific cases we wear nitrile gloves when handling the gel and a face shield when operating the light box.

    Risk to the Safety and Health of the General Public
    E. coli has a low, but not non-existent virulence. For this reason, we wear gloves and lab coats and wash our hands when entering and leaving the labs. This helps to reduce the risk to the safety and health of the general public.

    Risk to the Environment
    When disposing of things such as liquid cell culture, we sit them overnight in the fume cabinet with some bleach in them. We pour the liquid down the shoot in the fume cabinet on the next day and autoclave the containers. We do not pour hazardous chemicals down the sink. All of these action minimise the risks to the environment.

    Risks to Security Through Malicious Mis-Use
    Our project does not pose much of a risk in terms of malicious mis-use by others, but to try and prevent this we have key card access into both the building and the labs so that they cannot be accessed by the general public.

    Measures Taken to Reduce Risks
    We employ safe lab practices (i.e. wearing gloves and lab coats), dispose of waste appropriately, use a baceterium (E. coli) with a very low virulence, and have key card access to the labs.

     

  • 9. Risks of Our Project in the Future

    If our project is successful it will allow people to use the molecules we make as a platform technology for making disulphide bonded peptides. These peptides could potentially be used for harmful purposes. We don’t currently have any design features to reduce the risk of someone using our project for harmful purposes. In the future if it were deemed necessary we could potentially try to limit the types of amino acid residues or groups that could be added to the arms of our peptide by native chemical ligation.