Team:Sumbawagen/Safety

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                   <a href="#" class="dropdown-toggle" data-toggle="dropdown">Overviews <b class="caret"></b></a>
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                       <li><a href="#"> E-coney Project </a></li>
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                       <li><a href="https://2014.igem.org/Team:Sumbawagen/overviews/Econey_Project"> Econey Project </a></li>
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                   <a href="https://2014.igem.org/Team:Sumbawagen/Team" class="dropdown-toggle" data-toggle="dropdown">Project <b class="caret"></b></a>
                   <a href="https://2014.igem.org/Team:Sumbawagen/Team" class="dropdown-toggle" data-toggle="dropdown">Project <b class="caret"></b></a>
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                    <li class="nav-header">Lab</li>
 
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<li><a href="https://2014.igem.org/Team:Sumbawagen/project/vector">Vector Construction</a></li>
 
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<li><a href="https://2014.igem.org/Team:Sumbawagen/measure">Measure Cultivation</a></li>
 
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                    <li><a href="https://2014.igem.org/Team:Sumbawagen/Modeling">Modelling</a></li>
 
                    
                    
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<li><a href="https://2014.igem.org/Team:Sumbawagen/project/econey">Econey</a></li>
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                    <li class="nav-header">Software</li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/project/result">Result</a></li>
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                  <li><a href="https://2014.igem.org/Team:Sumbawagen/soft">E-Coney Soft</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/project/software">Software</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/parts">Parts</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Safety">Safety</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Attributions">Attribution</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/future_direction">Future Direction</a></li>                             
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                    <li class="nav-header">Attributions </li>
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                    <li class="nav-header">Notebook </li>
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                    <li><a href="https://2014.igem.org/Team:Sumbawagen/Notebook">Notebook</a></li>
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                   <a href="#" class="dropdown-toggle" data-toggle="dropdown">Notebook <b class="caret"></b></a>
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                     <li><a href="#">Our Biobricks</a></li>
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                    <li class="nav-header">Daily Notes</li>
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                    <li><a href="https://2014.igem.org/Team:Sumbawagen/Notebook">Policy & Practices</a></li>
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                    <li><a href="https://2014.igem.org/Team:Sumbawagen/Notebook2">Lab</a></li>
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                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Notebook/Protocol">Protocol</a></li>
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                 <li><a href="https://2014.igem.org/Team:Sumbawagen/Safety">Safety</a></li>
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                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Team">Meet the Team</a></li>
                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Team">Meet the Team</a></li>
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                    <li><a href="http:https://igem.org/Team.cgi">Team Information</a></li>
 
                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Team/Gallery">Gallery</a></li>
                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Team/Gallery">Gallery</a></li>
                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Team/Contact">Contact</a></li>
                     <li><a href="https://2014.igem.org/Team:Sumbawagen/Team/Contact">Contact</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/meet">Meet US Ambassador</a></li>
<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/meet">Meet US Ambassador</a></li>
<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/econey">@america</a></li>
<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/econey">@america</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/moyo">Moyo Festival</a></li>
<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/virtual">Virtual Outreach</a></li>
<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/virtual">Virtual Outreach</a></li>
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/Support">Responses and supports</a></li>
                                                            
                                                            
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           <h2><b>Safety</b> </h2>
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We aim to create an engineered E. coli which capable to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. In our project we create a novel circuit without producing any biosynthesis product. So, there’s no risk in commerce or industry. In this safety page, we divided our safety procedure into 3 general title, which are:<br>
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<ul style="padding-left:55px">
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<li>1. Safety Rules in Laboratory</li>
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<li>2. Safety Project</li>
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<li>3. Safety Form</li><br><br></ul>
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<center><center><img src="https://static.igem.org/mediawiki/2014/1/1b/Sumbawa_safety1.JPG"style="width:800px;height:400px;"/></center></center>
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<h2>1.Safety Rules in Laboratory</h2>
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<p>The basic rules in our laboratory are:</P>
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        <p> a. Everyone has been trained before participate the laboratory experiment. The training covered several topics, such as basic information of IGEM and synthetic biology, technique of DNA isolation and handling, cutting and joining of DNA, plasmid transformation, handling the synthetic biology waste and basic rules of laboratory safety.<br><br>
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b. Everyone who works in the laboratory should follow the basic standard of laboratory safety, such as wearing safety cloth (lab coat, rubber gloves, and surgery mask).<br><br>
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c. In order to minimize the contamination, we do the experiment in laminar air flow (clean bench), following the standard procedure.<br><br>
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d. Laboratory activities were done by the students under supervision of instructors.<br><br>
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        <p> Our project is to create an engineered E. coli which capable to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. In our project we create a novel circuit without producing any biosynthesis product, which not create a new risk in commerce or industry. In this safety page, we have divided our safety procedure into 3 general title, which are:<br><Br>
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<center><center><img src="https://static.igem.org/mediawiki/2014/1/1b/Sumbawa_safety1.JPG"style="width:500px;height:70px;"/></center></center>
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<center><center><img src="https://static.igem.org/mediawiki/2014/9/91/Safety_2.JPG"style="width:480px;height:359px;"/></center></center>
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           <div class="span12" style="text-align:justify"><p>The targeted modelling  work was essential for the development of our project as a whole, but the team also wanted to allow others to investigate, develop and test ideas related to our project and theirs too. We developed an interactive modelling tool based on NetLogo that allows the user to test a large variety of hypotheses connected to the production and export of PP1 and its function as a ToxiMop. This easy-to-use online programme gives instant visual as well as quantitative feedback to the user. </p></div>
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<h2>SECTION II: Protocol</h2>
 
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Introduction: There are no spectrophotometers – neither UV nor fluorescent – in Sumbawa island. Thus our project uses mobile phone camera to obtain data and quantify using customized software. Among fluorescent proteins, mRFP showed strong color visible to naked eye, thus we chose it as the report of measurement in our project. However, this report was made in the request of iGEM 2014 Committee using GFP to show the possibility on using our system for the Measurement Interlab Study. In this study we used different device/part although it has the same GFP gene, aside from which was already been designated by the Committee. We did it because the objective was to show whether GFP is measurable or not in our system.<br><br>
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<h2>2. Safety Project </h2>
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We are working with 2 types of E. coli strain; they are E. coli BL21 (DE3) and E. coli DH5α. Both of strain are classified in biosafety level 1 and including to risk group number 1 (low risk). It does not cause disease in healthy of adult humans and also does not have any additional safety precautions. We handle the organism safely by keep it into the safe place in our laboratory. We work in the lab where not all people have access to enter the lab.<br>
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Through this activity, people's understanding of Sumbawa honey is also increasing. We introduce to the public some information about Sumbawa honey such as how to distinguish between genuine and fake honey, a tool used to measure the levels of glucose in honey, until any product that can be produced made from honey. We hope this activity could expand the knowledge and society pride of Sumbawa honey. We also hope that our project will make people aware of the importance of maintaining the natural resource and pride of this region. When the awareness is growing, we believe the existence of honey as the pride of Sumbawa could be maintained.<br><br>
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<strong>Materials: <i>E. coli</i> BL21(DE3), and <i>E. coli</i> BL21(DE3) / BBa_I13522 </strong><br><br>
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Methods: Four glass tubes were prepared to cultivate overnight <i>E. coli</i> in 2 mL LB medium, in room temperature using shaker. The content of each tubes were as follow:<br><br>
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<li>Tube-1.  <i>E. coli</i> BL21(DE3) only. No antibiotics. Used as control.</li>
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        <p> Our project resulted biological waste. In order to dispose the biological wastes (for example used pipette tip), according the standard of laboratory safety, we collect them together in a certain box with label and decontaminated the autoclave before disposal or destroyed; the liquid waste such as cell culture and agar plate treated by decontamination with autoclave according to the laboratory standard operational procedures and dispose the material to the sanitary sewer system.<br><br>
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<li>Tube-2.   <i>E. coli</i> BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final).</li>
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We use lab coat, rubber gloves and surgery mask in order to minimize contamination of the experiment whether in the laboratory or workshop for the society.
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<li>Tube-3.  <i>E. coli</i> BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final); Auto induction reagents (Studier, Protein Expression and Purification
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41, 207-234, 2005).</li>
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<li>Tube-4. <i>E. coli</i> BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final); Glucose 200 mM.</li></ul><br><br>
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After one night of cultivation, tubes were collected, then photos were taken using pocket digital camera, and color of the medium were measured using free application available on Google Play named ColorMeter, an application developed by vistechprojects.com. The mobile phone used was Samsung GT-P5200 having 3.15 MP camera. The medium were centrifuged (6,000 rpm, 10 min.) in 1,5 ml polypropylene tube to obtain cell pellets, which were used to detect GFP fluorescence above a blue transilluminator (Pearl Biotech).<br><br>
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<h2>SECTION III: Results and Discussions</h2>
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In this study we used BBa_I13522, which already available in our lab, instead of BBa_I20260 which is designated as device no. 1 by Measurement Interlab Study Committee. As shown in Figure 1, both devices have the same RBSs, genes and terminators, except promoters. Because our intention here is to prove whether our system using mobile phone camera is suitable for GFP measurement or not, we use BBa_I13522 for convenient reason.<br>
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<center><img src="https://static.igem.org/mediawiki/2014/4/49/SBGI2.jpg"style="width:500px;height:70px;"/></center>
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<center><strong>Figure 1. Vector maps of BBa_I13522 (left) and BBa_I20260 (right)</strong><br><br></center>
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<center><center><img src="https://static.igem.org/mediawiki/2014/6/6f/Safety_3.JPG"style="width:480px;height:359px;"/></center></center>
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In Figure 2, it is showed that medium from 4 tubes showed similar color, although tube no. 3 has more green. Tube no. 1 and 2 are negative controls because they contain <i>E. coli</i> BL21(DE3) only, and <i>E. coli</i> BL21(DE3) / BBa_I13522 without expression reagent to induce GFP production, respectively. Tube no. 3 contains <i>E. coli</i> BL21(DE3) / BBa_I13522 with GFP was over expressed due to addition of auto induction reagent. On the other hand, glucose was added to tube no. 4 to repress possible leaky expression of GFP through the mechanism of catabolite repression.<br>
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<center><img src="https://static.igem.org/mediawiki/2014/a/a7/SBGI1.jpg"style="width:300px;height:175px;"/></center>
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<center><strong>Figure 2. Color of an overnight LB medium grew with <i>E. coli</i> under different condition.</strong></center>
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<center>1) <i>E. coli</i> BL21/DE3; 2) <i>E. coli</i> BL21 (DE3)/Bba_I13522; 3) <i>E. coli</i> BL21(DE3)/BBa_I13522 + autoinduction reagent; 4) <i>E. coli</i> BL21(DE3) + glucose 200 mM.<br></center></p>
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<br><p>ColorMeter program installed in mobile phone, detected RGB values, we then converted the values to luminance value to obtain only the brightness of color (http://www.scantips.com/lumin.html; accessed on Sep. 24th, 2014). The formula used is:</p>
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<p>Luminance value = (R x 0.30) + (G x 0.59) + (B x 0.11)</p>
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<h2>3. About our safety</h2>
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<ul style="padding-left:25px;">
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<li>Link to "Our Lab Form" <a href ="https://igem.org/safety/about_our_lab?team_id=1508">https://igem.org/safety/about_our_lab?team_id=1508:</a></li>
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<li>Link to “Safety Form” <a href ="https://igem.org/safety/safety_form?team_id=1508">https://igem.org/safety/safety_form?team_id=1508 </a></li>
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<li>Link to “Check-in” <a href ="https://igem.org/safety/check_in?team_id=1508">https://igem.org/safety/check_in?team_id=1508 </a></li>
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</ul>
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<br>Two measurements of RGB values of the tubes from different angles to correct possible lighting differences, and the calculation of luminance values are given in Table 1 and Figure 3 below.<br></p>
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<center><strong>Table 1. RGB and Luminance value obtained by ColorMeter program</strong></center>
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<center><img src="https://static.igem.org/mediawiki/2014/5/5f/Measurment_Interlab_Study_1.jpg"></center><br>
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<center><img src="https://static.igem.org/mediawiki/2013/c/cf/Grafik_Econey.png"></center>
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<center><strong>Figure 3. Luminance values of an overnight LB medium grew with <i>E. coli</i> under different condition. </strong></center>
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<center>1) <i>E. coli</i> BL21/DE3; 2) <i>E. coli BL21</i> (DE3)/Bba_I13522; 3)<i>E. coli</i> BL21(DE3)/BBa_I13522 + autoinduction reagent; 4)<i>E. coli</i> BL21(DE3) + glucose 200 mM.<br></center>
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<p>Expression of GFP in theory should make the color of medium brighter thus luminance value will be lower compared to E. coli which did not express GFP. Tube no. 1, and 4 has higher luminance value than tube no. 3, which is as expectation. But tube no. 2 showed low luminance value too. The reason may be because the color differences are too small (luminance value range < 5), thus accuracy of the results was low. In our project using mRFP (data not shown), the difference of luminance value between expressed and non-expressed mRFP were about 50.<br><br>
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Finally to validate the expression of GFP, we used the only equipment available in our Lab, which can visualize specifically fluorescent protein including GFP ((http://en.wikipedia.org/wiki/Green_fluorescent_protein; accessed on Sep. 24th, 2014). Two observations were made, in liquid and solid states. As shown in Figure 4 (top), observation of the culture medium showed that tube no. 3 has bright yellow color compared to other tubes. The color should come from GFP expressed by <i>E. coli</i> BL21(DE3)/BBa_I13522 cultured with auto induction reagent. However, the color was not very significant. Thus, cells were pelleted by centrifugation and once again observed. As shown in Figure 4 (bottom), tube no. 3 showed much bright yellow color compared to other tubes. This result concluded that this tube really contains<i>E. coli</i> BL21(DE3)/BBa_I13522 expressing GFP.<br><br>
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<center><img src="https://static.igem.org/mediawiki/2013/c/ce/Picture4.png"style="width:400px;height:200px;"/></center>
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<center><strong>Figure 4. Observation of GFP in liquid medium (top) and pellet (bottom) by transilluminator </strong></center>
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<center>1) <i>E. coli</i> BL21/DE3; 2) <i>E. coli</i> BL21 (DE3)/Bba_I13522; 3) <i>E. coli</i> BL21(DE3)/BBa_I13522 + auto induction reagent; 4) <i>E. coli</i> BL21(DE3) + glucose 200 mM</center><br><br>
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<p>In conclusion, our measurement system using mobile phone camera could not detect GFP expression in liquid medium because of small differences observed by naked eyes. Thus, we used mRFP instead for our iGEM project.</P>
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Latest revision as of 08:57, 19 February 2015

Team:Dundee/Team - 2013.igem.org

 

Team:Sumbawagen/Team

From 2014.igem.org

iGEM Sumbawagen 2014 · Econey

Sumbawagen

Safety

We aim to create an engineered E. coli which capable to measure the concentration of glucose in honey by calibrating the color of E. coli medium using android-based mobile phone. In our project we create a novel circuit without producing any biosynthesis product. So, there’s no risk in commerce or industry. In this safety page, we divided our safety procedure into 3 general title, which are:
  • 1. Safety Rules in Laboratory
  • 2. Safety Project
  • 3. Safety Form


1.Safety Rules in Laboratory

The basic rules in our laboratory are:

a. Everyone has been trained before participate the laboratory experiment. The training covered several topics, such as basic information of IGEM and synthetic biology, technique of DNA isolation and handling, cutting and joining of DNA, plasmid transformation, handling the synthetic biology waste and basic rules of laboratory safety.

b. Everyone who works in the laboratory should follow the basic standard of laboratory safety, such as wearing safety cloth (lab coat, rubber gloves, and surgery mask).

c. In order to minimize the contamination, we do the experiment in laminar air flow (clean bench), following the standard procedure.

d. Laboratory activities were done by the students under supervision of instructors.


2. Safety Project

We are working with 2 types of E. coli strain; they are E. coli BL21 (DE3) and E. coli DH5α. Both of strain are classified in biosafety level 1 and including to risk group number 1 (low risk). It does not cause disease in healthy of adult humans and also does not have any additional safety precautions. We handle the organism safely by keep it into the safe place in our laboratory. We work in the lab where not all people have access to enter the lab.

Our project resulted biological waste. In order to dispose the biological wastes (for example used pipette tip), according the standard of laboratory safety, we collect them together in a certain box with label and decontaminated the autoclave before disposal or destroyed; the liquid waste such as cell culture and agar plate treated by decontamination with autoclave according to the laboratory standard operational procedures and dispose the material to the sanitary sewer system.

We use lab coat, rubber gloves and surgery mask in order to minimize contamination of the experiment whether in the laboratory or workshop for the society.


3. About our safety

Sponsors




Retrieved from "https://2013.igem.org/Team:Dundee/Project/MopQuantifying"

Retrieved from "http://2014.igem.org/Team:Sumbawagen/Safety"