Difference between revisions of "Team:UofC Calgary/Safety"

 
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<a href="https://2016.igem.org/Team:UofC_Calgary/Model">Model</a>
 
<a href="https://2016.igem.org/Team:UofC_Calgary/Model">Model</a>
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<a href="https://2016.igem.org/Team:UofC_Calgary/Design">Applied Design</a>
 
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<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Parts Registry</a>
 
<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Parts Registry</a>
 
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                                                                <a href="https://2016.igem.org/Team:UofC_Calgary/Composite_Part">Composite Parts</a>
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<a href="https://2016.igem.org/Team:UofC_Calgary/HP/Gold">Gold</a>
 
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                                    <a href="https://2016.igem.org/Team:UofC_Calgary/Policy"> Policy Brief </a>
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                                    </li>
 
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<a href="https://2016.igem.org/Main_Page" data-toggle="modal" data-target="#login-form" class="c-btn-border-opacity-04 c-btn btn-no-focus c-btn-header btn btn-sm c-btn-border-1x c-btn-white c-btn-circle c-btn-sbold"><i class="icon-chemistry"></i> iGEM </a>
 
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<h2> Safety Considerations for Lab Work<h2>
+
<h1>Safety Considerations in the Lab<h1>
−
     <p class="c3"><span class="c1">All Principal Investigators, mentors, and undergraduate researchers were required to complete lab safety training and take safety courses developed by <a href="http://www.ucalgary.ca/safety/">Environment Health and Safety (EHS)</a> prior to working in the lab. The mandatory safety training included updated versions of the WHMIS course, the occupational health and safety course, the laboratory safety course, a hazard assessment course, an incident reporting and investigation course, a spill response course, a biosafety program course, a biosafety laboratory course, and a biosafety bloodborne pathogens course. The courses cover biological containment protocols, handling of hazardous materials such as liquid nitrogen, and disposal of waste as well as standard safety practices. All were required to take a test following each course, which certifies safe lab work under EHS Guidelines. All team members, advisors, and mentors received credit for each listed course and training program, and supervisors were present in the lab at all times to oversee undergraduate work.</span>
+
<h2><u>How we prepared for lab work</u></h2>
 +
     <p class="c3"><span class="c1">All Principal Investigators, mentors, and undergraduate researchers were required to complete lab safety training and safety courses developed by the University of Calgary's <a href="http://www.ucalgary.ca/safety/">Environment Health and Safety (EHS)</a> services prior to working in the lab. These mandatory safety training courses included courses on occupational health and safety, laboratory safety, hazard assessment, incident reporting and investigation, spill response, biosafety, bloodborne pathogens, and an updated versions of the WHMIS course. The courses cover biological containment protocols, handling of hazardous materials such as liquid nitrogen, and disposal of waste, as well as standard safety and laboratory practices. All required us to take a test following each course, which certified safe lab work under the EHS Guidelines. All team members, advisors, and mentors received credit for each course and training program listed, and supervisors were present in the lab at all times to oversee undergraduate work.</span>
 
     </p>
 
     </p>
 
     <p class="c2"><span class="c1"></span>
 
     <p class="c2"><span class="c1"></span>
 
     </p>
 
     </p>
−
    <ul >
+
     <p class="c3"><span class="c1">The University of Calgary has a university-wide <a href="http://www.ucalgary.ca/safety/ohsms-cor/health-safety-committees/biosafety-0">Biosafety Committee</a>, whose guidelines for safe biological laboratory practices were adhered to throughout the project. The team&rsquo;s lab benches and experimental plans were assessed and deemed safe to proceed with by this Biosafety Committee. The University's Environment Health and Safety (EHS) services provided additional training for individuals working with radiation and irradiated cells.</span>
−
     <p class="c3"><span class="c1">Our project utilized BSL1 cells (</span><span class="c8 c1">E.coli</span><span class="c1">&nbsp;and </span><span class="c8 c1">B.subtilis</span><span class="c1">) that have GRAS labelling, thus our main project did not require ethics approval by the review boards. The University of Calgary has a university-wide <a href="http://www.ucalgary.ca/research/researchers/ethics-compliance/biosafety">Biosafety Committee</a>, whose guidelines for safe biological laboratory practices were adhered to throughout the project.The team&rsquo;s lab benches and experimental plans were assessed and deemed safe to proceed with. The Environment Health and Safety (EHS) provided training and information on the hazards from the types of sources and each form of radiation. All researchers underwent safety training courses. The individuals who worked with irradiated cells received radiation safety training from the EHS.</span>
+
 
     </p>
 
     </p>
−
    <p class="c2"><span class="c1"></span>
+
  <p class="c2"><span class="c1"></span>
 
     </p>
 
     </p>
  
−
    <p class="c3">Our project utilized <span class="c1">Bacillus subtilis</span><span class="c1">&nbsp;and a commonly used lab-strain of </span><span class="c8 c1">Escherichia coli</span><span class="c1">&nbsp;TOP10 which are non-pathogenic and non-infectious as they are Biosafety Level 1 organisms (BSL-1). Thus the organisms were harmless to researchers. &nbsp;Additionally, the team worked with HCT116 cell lines, and 1BR3 cell lines which are Human colon carcinoma and Human skin fibroblast cell and are classified as Biosafety Level 2(BSL-2).The cell lines were received from completely anonymous donors. </span><span class="c4">We handled these cell lines at containment level 2 in accordance with the</span><span class="c1">&nbsp;Bloodborne Pathogens Standard and Biosafety Committee</span><span class="c4">&nbsp;guidelines.</span>
+
<p class="c3">Our project utilized <span class="c8 c1">Bacillus subtilis</span><span class="c1">&nbsp;and a commonly used lab-strain of </span><span class="c8 c1">Escherichia coli,</span><span class="c1">&nbsp;TOP10. Both are non-pathogenic and non-infectious, and are classified as Biosafety Level 1 organisms (BSL-1). Therefore, these organisms posed no significant risk to researchers. Since the BSL-1 cells (</span><span class="c8 c1">E. coli</span><span class="c1">&nbsp;and </span><span class="c8 c1">B. subtilis</span><span class="c1">) have GRAS labelling, the main cloning component of out project did not require ethics approval by review boards. Some team members worked with HCT116 and 1BR3 primary cell lines, which are human colon carcinoma and human skin fibroblast cell lines and are classified as Biosafety Level 2 (BSL-2).The cell lines were received from completely anonymous donors. </span><span class="c4">We handled these cell lines at containment level 2 in accordance with the</span><span class="c1">&nbsp;Bloodborne Pathogens Standard and Biosafety Committee</span><span class="c4">&nbsp;guidelines.</span>
 
         </p>
 
         </p>
−
<h2> Patch Design<h2>
+
 
−
     <ul>
+
<p><span></p></span>
−
         <li class="c0"><span>The circular design of the packet containing media and spores, encourages equal distribution of liquid pressure around the circumference. Since the packets are made up of flexible polyethylene material, this design is more advantageous when cases such as accidental bumping or &lsquo;squishing&rsquo; of the patch happen by distributing the force.</span>
+
     <h1><span>Safety Considerations for the Device</span></h1>
 +
    <p class="c3 c11"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 418.67px; height: 217.33px;"><img alt="" src="https://static.igem.org/mediawiki/2016/4/4a/T--UofC_Calgary--Syed00.jpg" style="width: 418.67px; height: 217.33px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span>
 +
    </p>
 +
    <h2><span><u>Structure of the Patch</u></span></h2>
 +
         <li class="c0"><span>The circular design of the packet containing media and <span class="c8 c1">B. subtilis</span> spores encourages equal distribution of liquid pressure around the circumference to prevent the patch from breaking open at the seams. The packets are made of a flexible polyethylene material, which is advantageous for distributing external forces against the patch, such as accidental bumping or squishing. This distribution of pressure prevents the material from rupturing due to accumulated forces along seams or corners.</span>
 
         </li>
 
         </li>
−
         <li class="c0"><span>Having a quadrilateral design can cause accumulation of pressure along the edges when a packet is accidentally squeezed. This would potentially rupture the material faster than having distributed pressure along its sides.</span>
+
         <li class="c0"><span>The fact that our patch is non-invasive and only present in the external part of the body promotes safety better than an implant.</span>
 
         </li>
 
         </li>
−
         <li class="c0"><span>The fact that our patch is also non-invasive and only present in the external part of the body promotes safety better than an implant.</span>
+
<h2><span><u>Choosing Patch Materials</u></span></h2>
 +
         <li class="c0"><span>All materials used in the patch are chemically and biologically compatible, which decreases the risk of an immune response in the user.</span>
 
         </li>
 
         </li>
−
    </ul>
+
         <li class="c0"><span>The backing layer of the patch is made up of a strong, flexible, and gas-permeable material. This allows users to move with ease and saves them the worry of the patch tearing apart. This layer also prevents bacteria from escaping.</span>
−
<h2>Choosing Patch Materials:</h2>
+
−
    <ul>
+
−
         <li class="c0"><span>All materials used in the patch is chemically compatible and biocompatible, which poses less risks of user immune response.</span>
+
 
         </li>
 
         </li>
−
         <li class="c0"><span>Backing layer: The backing layer is made up of strong, flexible, and gas permeable material. This will allow users to move with ease and saving them the worry of the patch tearing apart. This layer also prevents the bacteria from escaping.</span>
+
         <li class="c0"><span>The size controlling membrane is made up of polymers that prevent bacteria from flowing through the patch, but allow our peptide to pass. The bacteria will not come into contact with the skin, which protects the user from the possibility of infection or immune responses.</span>
 
         </li>
 
         </li>
−
         <li class="c0"><span>Size-controlling membrane: The size controlling membrane is made up of polymers that prevent bacteria from flowing through the patch, only allowing peptide to pass. The bacteria, therefore, will not cause infections as it is shielded from the skin.</span>
+
         <li class="c0"><span>The adhesive chosen  to affix the patch to the skin has high oxygen/gas permeability, causes low pain upon removal from sensitive skin, and promotes diffusivity of the drug. The adhesive has been tested by Dow Corning (the company that supplied us the adhesive), and it was found to cause no significant effects on test animals.</span>
 
         </li>
 
         </li>
−
         <li class="c0"><span>Adhesive: The adhesive chosen has high oxygen/gas permeability, causes low pain upon removal to sensitive skin, and promotes diffusivity of the drug. This has been tested by Dow Corning and causes no significant effects on test animals.</span>
+
         <li class="c0"><span>The release liner is peeled from the adhesive layer and disposed of when the patch is applied to the skin.</span>
 
         </li>
 
         </li>
−
        <li class="c0"><span>Release liner: The release liner is disposed after being peeled.</span>
+
<h2><span><u>Considering Human Use</u></span></h2>
 +
<h4>Advantages of a Patch</h4>
 +
<li class="c0"><span>Using a patch is preferable to many other drug delivery systems. A patch gives the user the freedom to continue a course of medications even with an upset stomach, as the drug will not need to go through the digestive system. It is less invasive than an implant or injection, and delivers the therapeutic agent continuously, meaning the dosage is always above the threshold required for a therapeutic effect, and the user does not need to worry about taking their medication at the same time each day.</span>
 
         </li>
 
         </li>
−
    </ul>
+
<h4> Possible Problems with the Patch</h4>
−
 
+
         <li class="c0"><span>Accidental breakage of the patch while attached to skin may cause infections if the area is not disinfected properly. The risk is increased if there are open wounds on the skin near the application area.</span>
−
<h2>Health factors:</h2>
+
−
    <ul>
+
−
         <li class="c0"><span>Using a patch gives users freedom to take medication even with an upset stomach, as the drug will not need to go through our digestive system.</span>
+
 
         </li>
 
         </li>
−
         <li class="c0"><span>Accidental breakage of the patch while attached to skin may cause infections if not disinfected properly.</span>
+
         <li class="c0"><span>Patch may cause severe harm if swallowed.</span>
 
         </li>
 
         </li>
−
         <li class="c0"><span>Patch may cause severe harm when swallowed.</span>
+
         <li class="c0"><span>Potential skin irritation may occur. However, our initial mouse trials have shown no signs of inflammation.</span>
 
         </li>
 
         </li>
−
         <li class="c0"><span>Potential skin irritation may occur. Our initial mouse trials however have shown no signs of inflammation.</span>
+
<h4><b><i>in vivo</i> Mouse Trials </b></h4>
 +
<p> <li class="c0"> With the mentorship of Dr. Jenne, the team wrote and submitted an extensive ethics application to the <a href=" http://www.ucalgary.ca/research/researchers/ethics-compliance/animal-use-protocols"> Health Sciences Animal Care Committee (HSACC)</a> at the University of Calgary. The application was evaluated on multiple grounds such as feasibility, morality, and precautions of the testing of our transdermal patch on mice models. The project idea, protocols, and the scientific significance of our transdermal patch were also reviewed. HSACC approved our project under the above mentioned parameters, as our protocols and procedures were deemed moral. The testing of our transdermal patch was carried out based on the approved flowcharts below: </li> </p><br>
 +
<br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2016/a/a3/T--UofC_Calgary--Mouse_Trials1.png" width="958" height="379"/>
 +
</center>
 +
<br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2016/9/94/T--UofC_Calgary--Mouse_Trials2.png" width="958" height="602"/>
 +
</center>
 +
<br>
 +
 
 +
<p> <li class="c0"> Since no member on the team had completed the Institutional Animal User Training Program (IAUTP) on mice handling and registered with the Institutional Research Information Services Soluion (IRISS), the mice handling work was completed by Dr. Jenne and members of his lab who had previous training and certification. Members of our iGEM team were present at all times during the testing of the transdermal patch in mice models but only as observers. </li> </p><br>
 +
<h2><span><u>Containment</u></span></h2>
 +
         <li class="c0"><span class="c1">Contamination is an issue both on earth and in space, as the bacteria in the patch may be able to infect users of the patch or colonize foreign planets should they be released. To prevent the bacteria from escaping from the patch, there are several engineering controls in place, such as the protective physical barriers that prevent bacteria from diffusing out of the patch, including the backing layer and the selective membrane.</span>
 +
      <span>Although our initial diffusion assays have shown that bacteria can diffuse through our semipermeable membrane, we have begun testing with other smaller filters including peptide dialysis membranes and 0.2 micron size filters. We have also had success with membranes used to filter-sterilize liquids.</span>
 +
        <li class="c0"><span>See pages 15 - 16 of the <a href="https://2016.igem.org/Team:UofC_Calgary/Design" style="padding-right:0px";>Device Applied Design Manual</a> to tackle contamination issues.</span>
 
         </li>
 
         </li>
−
    </ul>
+
<h2><span><u>Future Considerations for Patch Design</u></span></h2>
−
<p class="c2"><span class="c1"></span>
+
<li class="c0"><span class="c1">If we can determine a better membrane that prevents the diffusion of the bacteria, we can use a two layer semi permeable system where the first layer prevents the diffusion of the bacteria and a second layer which further filters BBI for diffusion.</span>
−
        <li class="c0"><span class="c1">Public safety: Auxotrophy using Threonine was integrated in </span><span class="c1 c8">Bacillus</span><span class="c1">&nbsp;making it unlikely it will survive outside of lab.</span>
+
 
         </li>
 
         </li>
−
<p class="c3"><span class="c1">The safety of the radioprotective patch was assessed by conducting </span><span class="c8 c1">in vivo</span><span class="c1">&nbsp;mouse trails to test the patch, safety of the bacteria and the protein BBI. The mouse trial protocol was followed and approved by the Health Sciences Animal Care Committee (HSACC) who review animal care and testing.</span>
+
<h2><span><u>Safe disposal:</u></span></h2>
 +
        <li class="c0"><span>See pages 11 and 12 of the <a href="https://2016.igem.org/Team:UofC_Calgary/Design";>Device Applied Design Manual</a> for disposal information.</span>
 +
        </li>
 +
    <p class="c2"><span></span>
 
     </p>
 
     </p>
−
    <ul>
+
<h1><span>Safety Considerations of Biobrick Parts</span></h1>
−
        <li class="c0"><span class="c1">Link to certificate/Approval: .</span>
+
<li class="c0"><span class="c1">Public safety: Threonine auxotrophy was employed where integrated cassettes containing flanking regions of homology for the </span><span class="c1 c8">B. subtilis thrC</span><span class="c1">&nbsp; gene were used. This knocked out threonine synthase in our chassis, making it unlikely it would survive outside of conditions where theronine is not supplied.</span>
 
         </li>
 
         </li>
−
    </ul>
+
<li class="c0"><span class="c1">Environmental safety: &nbsp;It&rsquo;s possible that our </span><span class="c8 c1">B. subtilis</span><span class="c1">&nbsp; transformed with mBBI could outcompete native microbes and potentially disrupt existing ecologies; however, the integrated mBBI genetic construct would not make a significant difference in the bacteria&rsquo;s survivability outside the lab. Given the nature of our project and safeguards we have implemented, none of our BioBricks confer unsafe risks to individuals nor the environment.</span>
−
<h3 class="c3" id="h.exhwv5k6w11g"><span class="c7">Prevent escape of the bacteria/contamination issues</span></h3>
+
−
    <ul>
+
−
        <li class="c0"><span>The backing layer and the size-controlling membrane prevents the bacteria from escaping outside of the patch and being released onto the skin, respectively.</span>
+
 
         </li>
 
         </li>
−
        <li class="c0"><span>Although our initial diffusion assays have shown that bacteria can diffuse through our semipermeable membrane, we have begun testing with other smaller filters including peptide dialysis membranes and 0.2 micron size filters.</span>
+
<h2><u>Future Considerations</u></h2>
−
        </li>
+
    <li class="c0"><span class="c1">We would engineer inducible kill switches that could eradicate the bacteria if need be. Additionally, integrating mBBI in various essential genes required for amino acid synthesis could provide more opportunities for auxotrophy, increasing the safety of using </span><span class="c8 c1">B. subtilis </span><span class="c1">in the device.</span>
−
        <li class="c0"><span>If we can determine a better membrane that prevents the diffusion of the bacteria, we can use a two layer semi permeable system where the first layer prevents the diffusion of the bacteria and a second layer which further filters BBI for diffusion.</span>
+
    </li>
−
        </li>
+
−
        <li class="c0"><span>See pages 15 - 16 of the Device Manual to tackle contamination issues.</span>
+
−
        </li>
+
−
    </ul>
+
−
    <ul class="c5 lst-kix_og921at2amv4-1 start">
+
−
        <li class="c3 c10"><span class="c1">Documentation of ethics approval from the animal studies: available upon request</span>
+
−
        </li>
+
−
    </ul>
+
 
<p class="c2"><span class="c1"></span>
 
<p class="c2"><span class="c1"></span>
−
<ul>
 
−
        <li class="c0"><span class="c1">The engineered organism could potentially disrupt existing ecologies and be difficult to contain; however, as mentioned previously, integrated BBI would not make a difference in the bacteria&rsquo;s survivability outside the lab, and we have taken care to ensure that the Threonine auxotrophy is fatal to engineered microbes. Namely, given the nature of our project and safeguards we have implemented, none of our BioBricks confer unsafe risks to individuals nor the environment.</span>
 
−
        </li>
 
−
    </ul>
 
−
    <p class="c2"><span class="c1"></span>
 
 
     </p>
 
     </p>
−
<h2> Future Considerations <h2>
+
         </p>
−
<p class="c3"><span class="c1">A kill switch in </span><span class="c8 c1">Bacillus</span><span class="c1">&nbsp;should be designed as engineering a kill switch into standardized plasmids could be useful for future iGEM competitions. Additionally, integrating BBI in multiple sites would give more auxotrophic sites, increasing the safety of using </span><span class="c8 c1">B.subtilis </span><span class="c1">in the device.</span>
+
−
    </p>
+
−
    <p class="c2"><span class="c1"></span>
+
−
    </p>
+
−
         <li class="c0"><span class="c1">Environmental safety: &nbsp;It&rsquo;s possible our </span><span class="c8 c1">Bacillus</span><span class="c1">&nbsp;incorporated with BBI could possibly outcompete native microbes, however the the bacteria&rsquo;s survivability outside the lab has not been increased enough to make a significant difference</span>
+
−
        </li>
+
−
    </ul>
+
−
    <p class="c2"><span class="c1"></span>
+
−
    </p>
+
−
    <ul>
+
−
        <li class="c0"><span class="c1">Contamination issues may be a problem in space if the bacteria were to escape. To protect against the event that our radioprotective patch causes contamination in space or on earth, we would engineer inducible kill switches that could eradicate the bacteria if need be. Additionally, engineering controls such as the protective physical barriers of the patch would prevent the outbreak of bacteria. Various safety mechanisms have been included in the device and they are listed below.</span>
+
−
        </li>
+
−
    </ul>
+
−
+
−
    <p class="c3"><span>(We are talking about the more ergonomic design here)</span>
+
−
    </p>
+
−
    <p class="c3 c11"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 418.67px; height: 217.33px;"><img alt="" src="https://static.igem.org/mediawiki/2016/4/4a/T--UofC_Calgary--Syed00.jpg" style="width: 418.67px; height: 217.33px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span>
+
−
    </p>
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    <h3 class="c3" id="h.x2scggi2lsww"><span class="c7">Design considerations to promote safety:</span></h3>
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        <li class="c0"><span>See pages 11 and 12 of the Device Manual for storage information.</span>
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    <p class="c3"><span>Device Manual:</span><span><a class="c6" href="https://www.google.com/url?q=https://drive.google.com/file/d/0B25REnfhyiNWM0hsNGtjRl9GWkk/view?usp%3Dsharing&amp;sa=D&amp;ust=1475971836674000&amp;usg=AFQjCNHOCLRY_e0DjvYJdj_DLRp2WkrxyQ">&nbsp;</a></span><span class="c12"><a class="c6" href="https://www.google.com/url?q=https://drive.google.com/file/d/0B25REnfhyiNWM0hsNGtjRl9GWkk/view?usp%3Dsharing&amp;sa=D&amp;ust=1475971836675000&amp;usg=AFQjCNEWFJhbi_JdcIdG27FiYIKXBQNl7w">https://drive.google.com/file/d/0B25REnfhyiNWM0hsNGtjRl9GWkk/view?usp=sharing</a></span>
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        <li class="c0"><span class="c1"><a href="https://2016.igem.org/Safety/About_Our_Lab?team_id=2008">About Our Lab</a></span>
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    <p class="c2"><span class="c12"><a class="c6" href="https://www.google.com/url?q=https://drive.google.com/file/d/0B25REnfhyiNWM0hsNGtjRl9GWkk/view?usp%3Dsharing&amp;sa=D&amp;ust=1475971836675000&amp;usg=AFQjCNEWFJhbi_JdcIdG27FiYIKXBQNl7w"></a></span>
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    <p class="c3"><span class="c1 c9">Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?</span>
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    <p class="c3"><span class="c1">None of the new Biobrick parts are thought to be hazardous as the proteins our genes code do not increase the pathogenicity of the bacteria.</span>
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    <p class="c3"><span class="c1 c9">Is there a local biosafety group, committee, or review board at your institution?</span>
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    <p class="c3"><span class="c1">Our project utilized BSL1 cells (</span><span class="c8 c1">E.coli</span><span class="c1">&nbsp;and </span><span class="c8 c1">B.subtilis</span><span class="c1">) that have GRAS labelling, thus our main project did not require ethics approval by the review boards. The University of Calgary has a university-wide <a href="http://www.ucalgary.ca/research/researchers/ethics-compliance/biosafety">Biosafety Committee</a>, whose guidelines for safe biological laboratory practices were adhered to throughout the project.The team&rsquo;s lab benches and experimental plans were assessed and deemed safe to proceed with. The Environment Health and Safety (EHS) provided training and information on the hazards from the types of sources and each form of radiation. All researchers underwent safety training courses. The individuals who worked with irradiated cells received radiation safety training from the EHS.</span>
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<li class="c3">Our project utilized <span class="c1">Bacillus subtilis</span><span class="c1">&nbsp;and a commonly used lab-strain of </span><span class="c8 c1">Escherichia coli</span><span class="c1">&nbsp;TOP10 which are non-pathogenic and non-infectious as they are Biosafety Level 1 organisms (BSL-1). Thus the organisms were harmless to researchers. &nbsp;Additionally, the team worked with HCT116 cell lines, and 1BR3 cell lines which are Human colon carcinoma and Human skin fibroblast cell and are classified as Biosafety Level 2(BSL-2).The cell lines were received from completely anonymous donors. </span><span class="c4">We handled these cell lines at containment level 2 in accordance with the</span><span class="c1">&nbsp;Bloodborne Pathogens Standard and Biosafety Committee</span><span class="c4">&nbsp;guidelines.</span>
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        <li class="c0"><span class="c1"><a href="https://2016.igem.org/Safety/About_Our_Project?team_id=2008">About Our Project</a></span>
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        <li class="c3 c10"><span class="c1">Extended Safety Form Pt 1</span>
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         <li class="c0"><span class="c1"><a href="https://2016.igem.org/Safety/Final_Safety_Form?team_id=2008">Final Safety Form</a></span>
 
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Our entire team received a full BioSafety education from the University of Calgary! This entailed going to classes to prepare for a final quiz that tested our ability to be safe in the lab. Several of our members also had radiation training and clearance to ensure that work done with radiation was safe!
 
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Latest revision as of 01:09, 20 October 2016

iGEM Calgary 2016

Safety

Safety Considerations in the Lab

How we prepared for lab work

All Principal Investigators, mentors, and undergraduate researchers were required to complete lab safety training and safety courses developed by the University of Calgary's Environment Health and Safety (EHS) services prior to working in the lab. These mandatory safety training courses included courses on occupational health and safety, laboratory safety, hazard assessment, incident reporting and investigation, spill response, biosafety, bloodborne pathogens, and an updated versions of the WHMIS course. The courses cover biological containment protocols, handling of hazardous materials such as liquid nitrogen, and disposal of waste, as well as standard safety and laboratory practices. All required us to take a test following each course, which certified safe lab work under the EHS Guidelines. All team members, advisors, and mentors received credit for each course and training program listed, and supervisors were present in the lab at all times to oversee undergraduate work.

The University of Calgary has a university-wide Biosafety Committee, whose guidelines for safe biological laboratory practices were adhered to throughout the project. The team’s lab benches and experimental plans were assessed and deemed safe to proceed with by this Biosafety Committee. The University's Environment Health and Safety (EHS) services provided additional training for individuals working with radiation and irradiated cells.

Our project utilized Bacillus subtilis and a commonly used lab-strain of Escherichia coli, TOP10. Both are non-pathogenic and non-infectious, and are classified as Biosafety Level 1 organisms (BSL-1). Therefore, these organisms posed no significant risk to researchers. Since the BSL-1 cells (E. coli and B. subtilis) have GRAS labelling, the main cloning component of out project did not require ethics approval by review boards. Some team members worked with HCT116 and 1BR3 primary cell lines, which are human colon carcinoma and human skin fibroblast cell lines and are classified as Biosafety Level 2 (BSL-2).The cell lines were received from completely anonymous donors. We handled these cell lines at containment level 2 in accordance with the Bloodborne Pathogens Standard and Biosafety Committee guidelines.

Safety Considerations for the Device

Structure of the Patch

  • The circular design of the packet containing media and B. subtilis spores encourages equal distribution of liquid pressure around the circumference to prevent the patch from breaking open at the seams. The packets are made of a flexible polyethylene material, which is advantageous for distributing external forces against the patch, such as accidental bumping or squishing. This distribution of pressure prevents the material from rupturing due to accumulated forces along seams or corners.
  • The fact that our patch is non-invasive and only present in the external part of the body promotes safety better than an implant.

    • Choosing Patch Materials

  • All materials used in the patch are chemically and biologically compatible, which decreases the risk of an immune response in the user.
  • The backing layer of the patch is made up of a strong, flexible, and gas-permeable material. This allows users to move with ease and saves them the worry of the patch tearing apart. This layer also prevents bacteria from escaping.
  • The size controlling membrane is made up of polymers that prevent bacteria from flowing through the patch, but allow our peptide to pass. The bacteria will not come into contact with the skin, which protects the user from the possibility of infection or immune responses.
  • The adhesive chosen to affix the patch to the skin has high oxygen/gas permeability, causes low pain upon removal from sensitive skin, and promotes diffusivity of the drug. The adhesive has been tested by Dow Corning (the company that supplied us the adhesive), and it was found to cause no significant effects on test animals.
  • The release liner is peeled from the adhesive layer and disposed of when the patch is applied to the skin.

    • Considering Human Use

    Advantages of a Patch

  • Using a patch is preferable to many other drug delivery systems. A patch gives the user the freedom to continue a course of medications even with an upset stomach, as the drug will not need to go through the digestive system. It is less invasive than an implant or injection, and delivers the therapeutic agent continuously, meaning the dosage is always above the threshold required for a therapeutic effect, and the user does not need to worry about taking their medication at the same time each day.

    • Possible Problems with the Patch

  • Accidental breakage of the patch while attached to skin may cause infections if the area is not disinfected properly. The risk is increased if there are open wounds on the skin near the application area.
  • Patch may cause severe harm if swallowed.
  • Potential skin irritation may occur. However, our initial mouse trials have shown no signs of inflammation.

    • in vivo Mouse Trials

  • With the mentorship of Dr. Jenne, the team wrote and submitted an extensive ethics application to the Health Sciences Animal Care Committee (HSACC) at the University of Calgary. The application was evaluated on multiple grounds such as feasibility, morality, and precautions of the testing of our transdermal patch on mice models. The project idea, protocols, and the scientific significance of our transdermal patch were also reviewed. HSACC approved our project under the above mentioned parameters, as our protocols and procedures were deemed moral. The testing of our transdermal patch was carried out based on the approved flowcharts below:





  • Since no member on the team had completed the Institutional Animal User Training Program (IAUTP) on mice handling and registered with the Institutional Research Information Services Soluion (IRISS), the mice handling work was completed by Dr. Jenne and members of his lab who had previous training and certification. Members of our iGEM team were present at all times during the testing of the transdermal patch in mice models but only as observers.


    • Containment

  • Contamination is an issue both on earth and in space, as the bacteria in the patch may be able to infect users of the patch or colonize foreign planets should they be released. To prevent the bacteria from escaping from the patch, there are several engineering controls in place, such as the protective physical barriers that prevent bacteria from diffusing out of the patch, including the backing layer and the selective membrane. Although our initial diffusion assays have shown that bacteria can diffuse through our semipermeable membrane, we have begun testing with other smaller filters including peptide dialysis membranes and 0.2 micron size filters. We have also had success with membranes used to filter-sterilize liquids.
  • See pages 15 - 16 of the Device Applied Design Manual to tackle contamination issues.

    • Future Considerations for Patch Design

  • If we can determine a better membrane that prevents the diffusion of the bacteria, we can use a two layer semi permeable system where the first layer prevents the diffusion of the bacteria and a second layer which further filters BBI for diffusion.

    • Safe disposal:

  • See pages 11 and 12 of the Device Applied Design Manual for disposal information.

    • Safety Considerations of Biobrick Parts

  • Public safety: Threonine auxotrophy was employed where integrated cassettes containing flanking regions of homology for the B. subtilis thrC  gene were used. This knocked out threonine synthase in our chassis, making it unlikely it would survive outside of conditions where theronine is not supplied.
  • Environmental safety:  It’s possible that our B. subtilis  transformed with mBBI could outcompete native microbes and potentially disrupt existing ecologies; however, the integrated mBBI genetic construct would not make a significant difference in the bacteria’s survivability outside the lab. Given the nature of our project and safeguards we have implemented, none of our BioBricks confer unsafe risks to individuals nor the environment.

    • Future Considerations

  • We would engineer inducible kill switches that could eradicate the bacteria if need be. Additionally, integrating mBBI in various essential genes required for amino acid synthesis could provide more opportunities for auxotrophy, increasing the safety of using B. subtilis in the device.

    • Safety Forms

  • About Our Lab
  • About Our Project
  • Final Safety Form

    • iGEM

    iGEM is an international competition promoting synthetic biology as a means to solve social, economic and humanitarian problems around the globe. The iGEM Jamboree is held in Boston annually. In 2016, over 300 teams are competing against each other.

    Latest Entries

    Fully Trained!

    Our entire team received a full BioSafety education from the University of Calgary! This entailed going to classes to prepare for a final quiz that tested our ability to be safe in the lab. Several of our members also had radiation training and clearance to ensure that work done with radiation was safe!

    Read More

    Latest Pictures

    View More

    Find us

    Located in Calgary, Alberta, Canada.

    • University of Calgary
    • igem.calgary@gmail.com

    Copyright © University of Calgary 2016.