Team:Sumbawagen/Safety

From 2014.igem.org

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<strong>1.Safety rules in laboratory</strong><br>
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<h2>1.Safety rules in laboratory<h2>
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           <div class="span12" style="text-align:justify"><p>c. In order to minimize the contamination, we done the experiment in laminar air flow (clean bench), following the standard procedure.<br>
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d.Laboratory  activity were done by the students under supervision of instructors.</p></div>
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<h2>2. Safety Project </h2>
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We are working with 2 type of E. coli strain, they are E. coli BL21 (DE3) and E. coli DH5α. Both of strain are classified in biosafety level 1 and categorized as risk group number 1 (low risk).  It does not cause disease in healthy 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 restricted laboratory where not all people have access to enter the laboratory.<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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<p>The basic rules in our laboratory are:</P>
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        <p> Biological wastes were produced as the by product of laboratory activity. In order to dispose the biological wastes (for example used pipette tip), according the standard of laboratory safety, we collect them together into 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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We use lab coat, rubber gloves and surgery mask in order to minimize contamination and hazard of the experiment wether in the laboratory or in the society (when the workshop activity taken place)
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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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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><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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<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/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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<h2>3. About our safety</h2>
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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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<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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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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Revision as of 14:06, 17 October 2014

Team:Dundee/Team - 2013.igem.org

 

Team:Sumbawagen/Team

From 2014.igem.org

iGEM Sumbawagen 2014 · Econey

Sumbawagen

Safety

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:
  • 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 who join the project has been trained and pass the evaluation before join to the laboratory experiment. The training covered several topics, such as basic information about IGEM and synthetic biology, technique of DNA isolation and handling, cutting and joining of DNA, plasmid transformation, handling of the synthetic biology waste and basic rules of laboratory safety

b. Everyone who working in the laboratory should following the basic standard of laboratory safety, such as wearing safety cloth (laboratory coat, rubber gloves, and surgery mask) while working in the experiment according to the risk level type.

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

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


2. Safety Project

We are working with 2 type of E. coli strain, they are E. coli BL21 (DE3) and E. coli DH5α. Both of strain are classified in biosafety level 1 and categorized as risk group number 1 (low risk). It does not cause disease in healthy 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 restricted laboratory where not all people have access to enter the laboratory.

The basic rules in our laboratory are:

Biological wastes were produced as the by product of laboratory activity. In order to dispose the biological wastes (for example used pipette tip), according the standard of laboratory safety, we collect them together into 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 and hazard of the experiment wether in the laboratory or in the society (when the workshop activity taken place)


3. About our safety

Sponsors




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

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