Team:XMU-China/Project Interlab

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/*Interlab*/
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<img id="Project_Interlab_title" class="Project_title" src="https://static.igem.org/mediawiki/2014/1/1f/Xmu_project_interlab_zwei.png"/>
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<br />
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<p style="text-align: center; line-height: 150%; font-size: 24px;">
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     <span style="font-size: 27px; font-weight: 700;">INTERLAB STUDY</span>
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    <span style="font-size: 1em;"><span style="font-size: 1em;"><span style="font-size: 24px;">Inter-Lab Study</span></span></span>
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</h1>
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<p style="font-size: 1em;">
<p style="font-size: 1em;">
     <span style="font-size: 1em;">The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.</span><span style="font-size: 1em;">&nbsp;</span>
     <span style="font-size: 1em;">The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.</span><span style="font-size: 1em;">&nbsp;</span>
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     <span style="font-size: 1em; font-weight: 700;">BBa_K1412716 </span>:<span style="font-size: 1em;">BBa_I20260</span> (J23101-B0032-E0040-B0015) in the pSB3K3 </span><span style="font-size: 1em;">backbone</span><span style="font-size: 1em;">.</span>
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     <span style="font-size: 1em; font-weight: 700;">BBa_K1412716</span>: <span style="font-size: 1em;">BBa_I20260</span> (J23101-B0032-E0040-B0015) in the pSB3K3 </span><span style="font-size: 1em;">backbone</span><span style="font-size: 1em;">.</span>
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     <span style="font-size: 1em; font-weight: 700;">BBa_K1412924</span>:<span style="font-size: 1em;"> <span>BBa_J23101</span> + <span>BBa_E0240</span> (B0032-E0040-B0015), in the pSB1C3 </span><span style="font-size: 1em;">backbone</span><span style="font-size: 1em;">.</span>
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     <span style="font-size: 1em; font-weight: 700;">BBa_K1412924</span>: <span style="font-size: 1em;"> <span>BBa_J23101</span> + <span>BBa_E0240</span> (B0032-E0040-B0015), in the pSB1C3 </span><span style="font-size: 1em;">backbone</span><span style="font-size: 1em;">.</span>
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     <span style="font-size: 1em; font-weight: 700;">BBa_K1412999</span>:<span style="font-size: 1em;">BBa_J23115</span> +<span> BBa_E0240</span> (B0032-E0040-B0015), in the pSB1C3 </span><span style="font-size: 1em;">backbone</span><span style="font-size: 1em;">.</span>
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     <span style="font-size: 1em; font-weight: 700;">BBa_K1412999</span>: <span style="font-size: 1em;">BBa_J23115</span> + <span>BBa_E0240</span> (B0032-E0040-B0015), in the pSB1C3 </span><span style="font-size: 1em;">backbone</span><span style="font-size: 1em;">.</span>
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     <span style="font-family: Wingdings;"></span><span style="font-family: Wingdings;"> </span><span style="font-size: 18px; font-weight: 700;">GFP generator with J23101</span><span style="font-size: 18px; font-weight: 700;"></span><span style="font-size: 18px; font-weight: 700;">J23115</span>
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     <span style="font-family: Wingdings;"></span><span style="font-family: Wingdings;"> </span><span style="font-size: 18px; font-weight: 700;">GFP generator with J23101</span><span style="font-size: 18px; font-weight: 700;">, </span><span style="font-size: 18px; font-weight: 700;">J23115</span>
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     <span style="font-size: 1em;">For device <span>BBa_J23115</span> + <span>BBa_E0240</span>, the promoter strength is the weakest, so that it’s difficult to observe green color in naked eyes.</span>
     <span style="font-size: 1em;">For device <span>BBa_J23115</span> + <span>BBa_E0240</span>, the promoter strength is the weakest, so that it’s difficult to observe green color in naked eyes.</span>
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                     <span style="font-size: 1em;font-family:Times New Roman; font-weight: 700;">Figure 1.</span><span style="font-size: 1em;"> GFP of different device under nature light. </span><span style="font-size: 1em; font-weight: 700;">1:</span><span style="font-size: 1em;"> K1412999 in DH5α; </span><span style="font-size: 1em; font-weight: 700;">2: </span><span style="font-size: 1em;">K1412716 in DH5α; </span><span style="font-size: 1em; font-weight: 700;">3:</span><span style="font-size: 1em;"> K1412716 (connect by us) in DH5α; </span><span style="font-size: 1em; font-weight: 700;">4: </span><span style="font-size: 1em;">K1412924 in DH5α.</span>
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                     <span style="font-size: 1em;font-family:Times New Roman; font-weight: 700;"><strong>Figure 1.</strong></span><span style="font-size: 1em;"> GFP of different device under nature light.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">1:</span><span style="font-size: 1em;"> <span>BBa_K1412999</span> in DH5α; </span><span style="font-size: 1em; font-weight: 700;">2:</span><span style="font-size: 1em;"> <span>BBa_K1412716</span> (reconstructed by us) in DH5α; </span><span style="font-size: 1em; font-weight: 700;">3:</span><span style="font-size: 1em;"> <span>BBa_K1412716</span> in DH5α; </span><span style="font-size: 1em; font-weight: 700;">4:</span><span style="font-size: 1em;"> <span>BBa_K1412924</span>  in DH5α.</span>
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     <span style="font-size: 1em;">The bacteria was cultured in the LB medium for 12 hrs at 37</span><span style="font-family: 微软雅黑; font-size: 1em;">℃</span><span style="font-size: 1em;"> shaking at 200 rpm in the table concentrator, then 1 ml bacterium solution was transferred into 1.5 ml centrifuge tube, and was centrifuged at 10000 rcf(g) for 1 min. The supernatant was discarded and the residuals was suspend by PBS. The solution was centrifuged again, and we got the bacteria precipitate as the picture shown in </span><span style="font-size: 1em; font-weight: 700;">Figure 1</span><span style="font-size: 1em;">. In which we can find that device K1412924 is greenish in natural light while device K1412716 (reconstructed by us) and K1412716 emit a canary yellow color, and device K1412999 show the color which is close to white.</span>
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     <span style="font-size: 1em;">The bacteria was cultured in the LB medium for 12 hrs at 37</span><span style="font-family: 微软雅黑; font-size: 1em;">℃</span><span style="font-size: 1em;"> shaking at 200 rpm in the table concentrator, then 1 ml bacterium solution was transferred into 1.5 ml centrifuge tube, and was centrifuged at 10000 rcf(g) for 1 min. The supernatant was discarded and the residuals was suspend by PBS. The solution was centrifuged again, and we got the bacteria precipitate as the picture shown in </span><span style="font-size: 1em; font-weight: 700;"><strong>Figure 1</strong></span><span style="font-size: 1em;">. In which we can find that device <span>BBa_K1412924</span> is greenish in natural light while device <span>BBa_K1412716</span> (reconstructed by us) and <span>BBa_K1412716</span> emit a canary yellow color, and device <span>BBa_K1412999</span> show the color which is close to white.</span>
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                     <span style="font-size: 1em;font-family:Times New Roman; font-weight: 700;">Figure 2.</span><span style="font-size: 1em;"> GFP of different devices under the UV-light.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">1:</span><span style="font-size: 1em;"> K1412924 in DH5α; </span><span style="font-size: 1em; font-weight: 700;">2:</span><span style="font-size: 1em;"> K1412716 (reconstructed by us) in DH5α; </span><span style="font-size: 1em; font-weight: 700;">3:</span><span style="font-size: 1em;"> K1412716 in DH5α; </span><span style="font-size: 1em; font-weight: 700;">4:</span><span style="font-size: 1em;"> K1412999 in DH5α.</span>
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                     <span style="font-size: 1em;font-family:Times New Roman; font-weight: 700;"><strong>Figure 2.</strong></span><span style="font-size: 1em;"> GFP of different devices under the UV-light.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">1:</span><span style="font-size: 1em;"> <span>BBa_K1412924</span> in DH5α; </span><span style="font-size: 1em; font-weight: 700;">2:</span><span style="font-size: 1em;"> <span>BBa_K1412716</span> (reconstructed by us) in DH5α; </span><span style="font-size: 1em; font-weight: 700;">3:</span><span style="font-size: 1em;"> <span>BBa_K1412716</span> in DH5α; </span><span style="font-size: 1em; font-weight: 700;">4:</span><span style="font-size: 1em;"> <span>BBa_K1412999</span> in DH5α.</span>
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     <span style="font-size: 1em;">Under UV-light, the bacterium precipitate above can be observed clearly that device K1412924 can emit strong green fluorescence, while devices K1412716 (reconstructed by us) and K1412716 have weaker green fluorescence, and the green fluorescence from device K1412999 is the weakest so that we can’t even observe</span><span style="font-size: 1em;"> a green pixel.</span>
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     <span style="font-size: 1em;">Under UV-light, the bacterium precipitate above can be observed clearly that device <span>BBa_K1412924</span> can emit strong green fluorescence, while devices <span>BBa_K1412716</span> (reconstructed by us) and <span>BBa_K1412716</span> have weaker green fluorescence, and the green fluorescence from device <span>BBa_K1412999</span> is the weakest so that we can’t even observe</span><span style="font-size: 1em;"> a green pixel.</span>
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<table style="border: currentColor; border-image: none; width: 800px; margin-right: auto; margin-left: auto; border-collapse: collapse; styleName: Normal Table; border-insideH: 1px none #000000; border-insideV: 1px none #000000; cellpadding: 0px 7px 0px 7px;font-family:Times New Roman;line-height:1;font-family:times new roman;">
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<p style="font-size:14px;">
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    <span style="font-weight:700;font-size:18px;">Devices</span><span style="font-weight:700;font-size:18px;"> </span><span style="font-weight:700;font-size:18px;">Verification</span>
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</p>
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<table style="styleName:Table Grid;margin-left:0px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;border-insideH:1px solid #000000;border-insideV:1px solid #000000;cellpadding:0px 7px 0px 7px;styleName:Normal Table;margin-left:0px;cellpadding:0px 7px 0px 7px;width:604px;margin-left:auto;margin-right:auto;border-collapse:collapse;border:none;font-family:times new roman;">
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                 <p style="font-size:14px;">
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                     <img width="297" height="384" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/9/94/%E8%83%B6%E5%9B%BE%E9%AA%8C%E8%AF%811.png"/>
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                     <img width="297" height="384" src="https://static.igem.org/mediawiki/parts/9/94/%E8%83%B6%E5%9B%BE%E9%AA%8C%E8%AF%811.png" style="font-size:14px;"/>
                 </p>
                 </p>
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             <td style="border-width: 1px 1px 1px medium; border-color: rgb(0, 0, 0) rgb(0, 0, 0) rgb(0, 0, 0) currentColor; padding: 0px 7px; width: 331px; vertical-align: top;">
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             <td style="width:293px;vertical-align:top;padding:0px 7px 0px 7px;border-top:1px solid #000000;border-left:none;border-bottom:1px solid #000000;border-right:1px solid #000000;">
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                <p style="font-size: 1em;">
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                <p style="font-size:14px;margin-left:28px;">
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                    <span style="font-size: 1em; font-weight: 700;font-family:Times New Roman;">Figure 3.</span><span style="font-size: 1em;"> Enzyme digestion verification of devices <span>BBa_J23115</span> + <span>BBa_E0240</span> and <span>BBa_I20260</span>.</span>
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                    <br/>
                 </p>
                 </p>
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                 <p style="font-size: 1em;">
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                 <p style="font-size:14px;margin-left:28px;">
-
                    <span style="font-family:Times New Roman;font-size: 1em;">Results and discussion: For device <span>BBa_I20260</span>, it’s very abnormal that we get puzzling results with double enzymes digestion by Xba I and Pst I. The target segment seems vanished. However, if we use </span><span style="font-size: 1em;">EcoR I</span><span style="font-size: 1em;"> and Pst I instead, we find that the segments, whose backbone is PSB3k3, generated by mono-restriction digestion is about 1000bp longer than that generated by double restriction enzyme digestion, and the devices have been verified by DNA sequencing. We can also get segments slightly shorter than 1000bp which generated by double digestion, and those segments are highlighted by red frames. So we confirm that device <span>BBa_I20260</span> is correct. We took our actual measurement with the reconstructed device.</span>
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                    <br/>
                 </p>
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                 <p style="text-indent: -28px; font-size: 1em; margin-left: 28px;">
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                 <p style="font-size:14px;margin-left:28px;">
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                     <span style="font-size: 1em;">1.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">1kb Marker;</span>
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                     <br/>
                 </p>
                 </p>
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                 <p style="text-indent: -28px; font-size: 1em; margin-left: 28px;">
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                 <p style="font-size:14px;margin-left:28px;text-indent:-28px;font-family:times new roman;">
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                    <span style="font-size: 1em;">2.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">Double restriction enzyme digestion (</span><span style="font-size: 1em;">EcoRI</span><span style="font-size: 1em;"> and PstI) with device <span>BBa_J23115</span> + <span>BBa_E0240</span> (constructed by XMU-China).</span>
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                    <span style="font-size:14px;">1.</span><span style="font-size:14px;"></span><span style="font-size:14px;">1kb Marker;</span>
                 </p>
                 </p>
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                 <p style="text-indent: -28px; font-size: 1em; margin-left: 28px;">
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                 <p style="font-size:14px;margin-left:28px;text-indent:-28px;">
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                     <span style="font-size: 1em;">3.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">Mono-restriction enzyme digestion (PstI) with device <span>BBa_J23115</span> + <span>BBa_E0240</span> (constructed by XMU-China).</span>
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                     <span style="font-size:14px;">2.</span><span style="font-size:14px;"></span><span style="font-size:14px;">Double restriction enzyme digestion (</span><span style="font-size:14px;"><i>EcoR</i> I</span><span style="font-size:14px;"> and <i>Pst</i> I) with device <span>BBa_J23115</span> + <span>BBa_E0240</span> (constructed by us).</span>
                 </p>
                 </p>
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                 <p style="text-indent: -28px; font-size: 1em; margin-left: 28px;">
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                 <p style="font-size:14px;margin-left:28px;text-indent:-28px;">
-
                     <span style="font-size: 1em;">4.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">Double restriction enzyme digestion (EcoRI and PstI) with device <span>BBa_I20260</span> (</span><span style="font-size: 1em;">reconstructed</span><span style="font-size: 1em;"> by XMU-China).</span>
+
                     <span style="font-size:14px;">3.</span><span style="font-size:14px;"></span><span style="font-size:14px;">Mono-restriction enzyme digestion (<i>Pst</i> I) with device <span>BBa_J23115</span> + <span>BBa_E0240</span> (constructed by XMU-China).</span>
                 </p>
                 </p>
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                 <p style="text-indent: -28px; font-size: 1em; margin-left: 28px;">
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                 <p style="font-size:14px;margin-left:28px;text-indent:-28px;">
-
                     <span style="font-size: 1em;">5.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">Mono-restriction enzyme digestion (PstI) with device <span>BBa_I20260</span> (reconstructed by XMU-China).</span>
+
                    <span style="font-size:14px;">4.</span><span style="font-size:14px;"></span><span style="font-size:14px;">Double restriction enzyme digestion (<i>EcoR</i> I and <i>Pst</i> I) with device <span>BBa_I20260</span> (</span><span style="font-size:14px;">reconstructed</span><span style="font-size:14px;"> by us).</span>
 +
                </p>
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                <p style="font-size:14px;margin-left:28px;text-indent:-28px;">
 +
                     <span style="font-size:14px;">5.</span><span style="font-size:14px;"></span><span style="font-size:14px;">Mono-restriction enzyme digestion (<i>Pst</i> I) with device <span>BBa_I20260</span> (reconstructed by us).</span>
                 </p>
                 </p>
             </td>
             </td>
         </tr>
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             <td colspan="2" style="width:604px;vertical-align:top;padding:0px 7px 0px 7px;border-top:none;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;">
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             <td style="border: currentColor; border-image: none; width: 331px;"></td>
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                <p style="font-size:14px;font-family:Times New Roman;">
 +
                    <span style="font-weight:700;font-size:14px;"><strong>Figure 3.</strong></span><span style="font-size:14px;"> Enzyme digestion verification of devices <span>BBa_J23115</span> + <span>BBa_E0240</span> and <span>BBa_I20260</span>.</span><span style="font-size:14px;"> </span>
 +
                </p>
 +
                <p style="font-size:14px;">
 +
                    <span style="font-size:14px;">Results and discussion: For device <span>BBa_I20260</span>, it’s very abnormal that we get puzzling results with double enzymes digestion by <i>Xba</i> I and <i>Pst</i> I. The target segment seems vanished. However, if we use </span><span style="font-size:14px;"><i>EcoR</i> I</span><span style="font-size:14px;"> and <i>Pst</i> I instead, we find that the segments, whose backbone is PSB3k3, generated by mono-restriction digestion is about 1000bp longer than that generated by double restriction enzyme digestion, and the devices have been verified by DNA sequencing. We can also get segments slightly shorter than 1000bp which generated by double digestion, and those segments are highlighted by red frames. So we confirm that device <span>BBa_I20260</span> is correct. We took our actual measurement with the reconstructed device.</span>
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                </p>
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             <td style="width:312px;border:none;"></td>
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<p style="font-size: 1em;">
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<p style="font-size:14px;">
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     &nbsp;
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     <br/>
</p>
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<p style="font-size:14px;">
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</p>
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<table style="styleName:Table Grid;margin-left:0px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;border-insideH:1px solid #000000;border-insideV:1px solid #000000;cellpadding:0px 7px 0px 7px;styleName:Normal Table;margin-left:0px;cellpadding:0px 7px 0px 7px;width:586px;margin-left:auto;margin-right:auto;border-collapse:collapse;border:none;font-family:times new roman;">
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             <td style="width:312px;vertical-align:top;padding:0px 7px 0px 7px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;">
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                 <p style="font-size: 1em;">
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                 <p style="font-size:14px;">
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                     <img width="324" height="395" src="https://static.igem.org/mediawiki/parts/0/0f/%E8%83%B6%E5%9B%BE%E9%AA%8C%E8%AF%812.png"/>
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                     <img width="324" height="395" src="https://static.igem.org/mediawiki/parts/0/0f/%E8%83%B6%E5%9B%BE%E9%AA%8C%E8%AF%812.png" style="font-size:14px;"/>
                 </p>
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                <p style="font-size: 1em;font-family:Times New Roman;">
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                <p style="font-size:14px;margin-left:24px;">
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                    <span style="font-size: 1em; font-weight: 700;">Figure 4.</span><span style="font-size: 1em;"> Enzyme digestion verification of devices <span>BBa_J23101</span> + <span>BBa_E0240</span></span>
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                    <br/>
                 </p>
                 </p>
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                 <p style="text-indent: -24px; font-size: 1em; margin-left: 24px;">
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                 <p style="font-size:14px;margin-left:24px;">
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                     <span style="font-size: 1em;">1.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">100bp Marker.</span>
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                     <br/>
                 </p>
                 </p>
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                 <p style="text-indent: -24px; font-size: 1em; margin-left: 24px;">
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                 <p style="font-size:14px;margin-left:24px;">
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                     <span style="font-size: 1em;">2.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;"><span>BBa_K1412924</span> with double enzymes (XbaI and PstI) digestion.</span>
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                     <br/>
                 </p>
                 </p>
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                 <p style="text-indent: -24px; font-size: 1em; margin-left: 24px;">
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                 <p style="font-size:14px;margin-left:24px;">
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                     <span style="font-size: 1em;">3.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">1kb Marker.</span>
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                     <br/>
                 </p>
                 </p>
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                 <p style="font-size: 1em;">
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                 <p style="font-size:14px;margin-left:24px;">
-
                     &nbsp;
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                     <br/>
                 </p>
                 </p>
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                 <p style="font-size: 1em;">
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                 <p style="font-size:14px;margin-left:24px;text-indent:-24px;">
-
                     <span style="font-size: 1em;">As double restriction enzyme digestion generates two target segments, we confirm that device <span>BBa_J23101</span> + <span>BBa_E0240</span> is constructed correctly.</span>
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                     <span style="font-size:14px;">1.</span><span style="font-size:14px;"></span><span style="font-size:14px;">100 bp Marker.</span>
 +
                </p>
 +
                <p style="font-size:14px;margin-left:24px;text-indent:-24px;">
 +
                    <span style="font-size:14px;">2.</span><span style="font-size:14px;"></span><span>BBa_K1412924</span> with double enzymes (<i>Xba</i> I and <i>Pst</i> I) digestion.
 +
                </p>
 +
                <p style="font-size:14px;margin-left:24px;text-indent:-24px;">
 +
                    <span style="font-size:14px;">3.</span><span style="font-size:14px;"></span><span style="font-size:14px;">1kb Marker.</span>
 +
                </p>
 +
                <p style="font-size:14px;">
 +
                    <br/>
                 </p>
                 </p>
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             <td style="border: currentColor; border-image: none; width: 304px;"></td>
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                <p style="font-size:14px;font-family:Times New Roman;">
 +
                    <span style="font-weight:700;font-size:14px;"><strong>Figure 4.</strong></span><span style="font-size:14px;"> Enzyme digestion verification of devices <span>BBa_J23101</span> + <span>BBa_E0240</span></span>
 +
                </p>
 +
                <p style="font-size:14px;">
 +
                    <span style="font-size:14px;">As double restriction enzyme digestion generates two target segments, we confirm that device <span>BBa_J23101</span> + <span>BBa_E0240</span> is constructed correctly.</span>
 +
                </p>
 +
            </td>
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        </tr>
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        <tr style="height:0;">
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             <td style="width:312px;border:none;"></td>
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            <td style="width:273px;border:none;"></td>
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     </tbody>
     </tbody>
</table>
</table>
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<p style="text-align: justify; text-indent: -28px; font-size: 18px; margin-left: 28px; styleName: Normal;">
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<p style="font-size:18px;">
-
     <span style="font-family: Wingdings;"></span><span style="font-family: Wingdings;"> </span><span style="font-size: 18px; font-weight: 700;">Protocol</span>
+
     <br/>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:18px;margin-left:24px;">
-
     <span style="font-size: 1em;">1. Transformed <span>BBa_K1412924</span> into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37</span><span style="font-family: 宋体; font-size: 1em;">℃</span><span style="font-size: 1em;">.</span>
+
     <span style="font-weight:700;font-size:18px;">Protocol</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">2. Inoculate a 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate.</span>
+
     <span style="font-size:14px;">1. Transformed <span>BBa_K1412924</span> into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37</span><span style="font-family:宋体;font-size:14px;">℃</span><span style="font-size:14px;">.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">3. Cultures were grown in conical flask for 16 hrs at 37</span><span style="font-family: 宋体; font-size: 1em;">℃</span><span style="font-size: 1em;"> with shaking at 200 rpm in the table concentrator.</span>
+
     <span style="font-size:14px;">2. Inoculate a 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">4. Cultures were diluted 1:100 into three 20 ml fresh LB medium and grown for 3 hrs at 37</span><span style="font-family: 宋体; font-size: 1em;">℃</span><span style="font-size: 1em;"> with shaking at 200 rpm in the table concentrator.</span>
+
     <span style="font-size:14px;">3. Cultures were grown in conical flask for 16 hrs at 37</span><span style="font-family:宋体;font-size:14px;">℃</span><span style="font-size:14px;"> with shaking at 200 rpm in the table concentrator.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">5. Then transfered 650 μl of the culture to a 1.5 ml centrifuge tube, centrifuged and washed twice with phosphate-buffered saline (PBS, pH 7.4) to minimize the background fluorescence from the medium.</span>
+
     <span style="font-size:14px;">4. Cultures were diluted 1:100 into three 20 ml fresh LB medium and grown for 3 hrs at 37</span><span style="font-family:宋体;font-size:14px;">℃</span><span style="font-size:14px;"> with shaking at 200 rpm in the table concentrator.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">6. The washed cells were suspended in PBS and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements.</span>
+
     <span style="font-size:14px;">5. Then transfered 650 μl of the culture to a 1.5 ml centrifuge tube, centrifuged and washed twice with phosphate-buffered saline (PBS, pH 7.4) to minimize the background fluorescence from the medium.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">7. Measure the fluorescence and absorbance:</span>
+
     <span style="font-size:14px;">6. The washed cells were suspended in PBS and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">(1)Fluorescence:</span>
+
     <span style="font-size:14px;">7. Measure the fluorescence and absorbance:</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">Device: SpectraMax+M5 microplate reader, 96-well plates.</span>
+
     <span style="font-size:14px;">(1)Fluorescence:</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.</span>
+
     <span style="font-size:14px;">Device: SpectraMax+M5 microplate reader, 96-well plates.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">(2)OD600 (optical density at 600 nm):</span>
+
     <span style="font-size:14px;">Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">Device: SpectraMax+M5 microplate reader, 96-well plates.</span>
+
     <span style="font-size:14px;">(2)OD600 (optical density at 600 nm):</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">Wavelengths: 600 nm absorption.</span>
+
     <span style="font-size:14px;">Device: SpectraMax+M5 microplate reader, 96-well plates.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     <span style="font-size: 1em;">8. Measure every 30 minutes in the next 4 hrs.</span>
+
     <span style="font-size:14px;">Wavelengths: 600 nm absorption.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;margin-left:24px;">
-
     &nbsp;
+
     <span style="font-size:14px;">8. Measure every 30 minutes in the next 4 hrs.</span>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:14px;">
-
     &nbsp;
+
     <br/>
</p>
</p>
-
<p style="font-size: 1em; margin-left: 24px;">
+
<p style="font-size:18px;margin-left:0.02em;">
-
     &nbsp;
+
     <span style="font-family:Wingdings;"></span><span style="font-family:Wingdings;"></span><span style="font-weight:700;font-size:18px;">Experimental data</span>
</p>
</p>
-
<p style="font-size: 18px; margin-left: 0.02em;">
+
<p style="font-size:14px;">
-
     <span style="font-family: Wingdings;"></span><span style="font-family: Wingdings;"> </span><span style="font-size: 18px; font-weight: 700;">Experimental data</span>
+
     <span style="font-size:14px;">Before the measurement, in order to set date in suitable range we estimated an appropriate concentration range by diluting the bacterium culture 2 times with PBS buffer. The values of PBS, the background, were subtracted during the data processing. And the data was removed when their deviations were too large. Then the remained data were doubled to get the final values to plot graph.</span>
</p>
</p>
-
<p style="font-size: 1em;">
+
<p style="font-size:14px;margin-left:48px;text-indent:-24px;">
-
     <span style="font-size: 1em;">Before the measurement, in order to set date in suitable range we estimated an appropriate concentration range by diluting the bacterium culture 2 times with PBS buffer. The values of PBS, the background, were subtracted during the data processing. And the data was removed when their deviations were too large. Then the remained data were doubled to get the final values to plot graph.</span>
+
     <span style="font-size:14px;">1.</span><span style="font-size:14px;"></span><br style="font-size:14px;"/>
</p>
</p>
-
<p style="font-size: 1em;">
+
<table style="styleName:Table Grid;margin-left:0px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;border-insideH:1px solid #000000;border-insideV:1px solid #000000;cellpadding:0px 7px 0px 7px;styleName:Normal Table;margin-left:0px;cellpadding:0px 7px 0px 7px;width:519px;margin-left:auto;margin-right:auto;border-top:1px none #000000;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;border-insideH:1px none #000000;border-insideV:1px none #000000;border-collapse:collapse;border:none;font-family:times new roman;">
-
    <span style="font-size: 1em; font-weight: 700;">All of our date are showing in graphs below. And our raw date are listed in appendix files.</span>
+
-
</p>
+
-
<p style="text-indent: -24px; font-size: 1em; margin-left: 48px;">
+
-
    <span style="font-size: 1em;">1.</span><span style="font-size: 1em;"> </span><br style="font-size: 1em;"/>
+
-
</p>
+
-
<table style="border: currentColor; border-image: none; width: 605px; margin-right: auto; margin-left: auto; border-collapse: collapse; styleName: Normal Table; border-insideH: 1px none #000000; border-insideV: 1px none #000000; cellpadding: 0px 7px 0px 7px;">
+
     <tbody>
     <tbody>
         <tr class="firstRow">
         <tr class="firstRow">
-
             <td style="padding: 0px 7px; border: 1px rgb(0, 0, 0); width: 605px; vertical-align: top;" colspan="2">
+
             <td style="width:519px;vertical-align:top;padding:0px 7px 0px 7px;border-top:1px none #000000;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;">
-
                 <p style="font-size: 1em;">
+
                 <p style="font-size:14px;">
-
                     <img width="590" height="340" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/e/ee/OD-T_924%2C999%2C716.png"/>
+
                     <img width="514.16" height="473.14" src="https://static.igem.org/mediawiki/2014/0/06/Xmu-interlab-007.png" style="font-size:14px;"/>
                 </p>
                 </p>
             </td>
             </td>
         </tr>
         </tr>
-
         <tr style="height: 227px;">
+
         <tr style="height:93px;">
-
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 605px; vertical-align: top;">
+
             <td style="width:519px;vertical-align:top;padding:0px 7px 0px 7px;border-top:none;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;">
-
                 <p style="font-size: 1em;">
+
                 <p style="font-size:14px;font-family:times new roman;">
-
                     <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/b/b6/OD-T_924.png"/>
+
                     <span style="font-weight:700;font-size:14px;"><strong>Figure</strong></span><span style="font-size:14px;"> </span><span style="font-weight:700;font-size:14px;"><strong>5.</strong></span><span style="font-size:14px;"> T</span><span style="font-size:14px;">he plot of OD versus time</span><span style="font-size:14px;">. Top row</span><span style="font-size:14px;"> are the plots of OD ver</span><span style="font-size:14px;">sus time for each single device</span><span style="font-size:14px;">. </span><span style="font-size:14px;">Bottom row are combination data of all three devices with error bar. </span><span style="font-size:14px;">We can conclude that the growth rate is </span><span style="font-size:14px;">getting</span><span style="font-size:14px;"> </span><span style="font-size:14px;">slower</span><span style="font-size:14px;"> with time</span><span style="font-size:14px;"> increasing</span><span style="font-size:14px;">. We measured </span><span style="font-size:14px;">each device with three </span><span style="font-size:14px;">samples</span><span style="font-size:14px;"> for three times</span><span style="font-size:14px;"> </span><span style="font-size:14px;">parallelly, and we make sure</span><span style="font-size:14px;"> that the reproducibility of the data is acceptable. </span><span style="font-size:14px;">Comparing</span><span style="font-size:14px;"> the plot of OD versus time with each other, we can </span><span style="font-size:14px;">see</span><span style="font-size:14px;"> that their growth rate are almost </span><span style="font-size:14px;">the same</span><span style="font-size:14px;">.</span>
-
                </p>
+
-
            </td>
+
-
            <td style="border-width: medium 1px 1px medium; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0) currentColor; padding: 0px 7px; width: 605px; vertical-align: top;">
+
-
                <p style="font-size: 1em;font-family:Times New Roman;">
+
-
                    <span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">1</span><span style="font-size: 1em;"> is the combination of </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3</span><span style="font-size: 1em;"> and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;">. </span><span style="font-size: 1em; font-weight: 700;">Figure 2, 3</span><span style="font-size: 1em;"> and</span><span style="font-size: 1em; font-weight: 700;"> 4</span><span style="font-size: 1em;"> are the plots of OD versus time for each single devices.</span><span style="font-size: 1em;"> We can conclude that the growth rate of bacteria is become lower with time. We measured the samples three times parallelly, and we can know that the reproducibility of the data is acceptable. When we compare </span><span style="font-size: 1em;">the plot of OD versus time with each other</span><span style="font-size: 1em;">, we can get that their growth rate are almost equal.</span>
+
                 </p>
                 </p>
             </td>
             </td>
         </tr>
         </tr>
-
         <tr style="height: 242px;">
+
         <tr style="height:0;">
-
            <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 605px; vertical-align: top;">
+
             <td style="width:519px;border:none;"></td>
-
                <p style="font-size: 1em;">
+
-
                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/8/86/OD-T_999.png"/>
+
-
                </p>
+
-
            </td>
+
-
             <td style="border-width: medium 1px 1px medium; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0) currentColor; padding: 0px 7px; width: 605px; vertical-align: top;">
+
-
                <p style="font-size: 1em;">
+
-
                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/6/60/OD-T_716.png"/>
+
-
                </p>
+
-
            </td>
+
-
        </tr>
+
-
        <tr style="height: 0px;">
+
-
            <td style="border: currentColor; border-image: none; width: 303px;"></td>
+
-
            <td style="border: currentColor; border-image: none; width: 302px;"></td>
+
         </tr>
         </tr>
     </tbody>
     </tbody>
</table>
</table>
-
<p style="font-size: 1em;">
+
<p style="font-size:14px;">
-
     &nbsp;
+
     <br/>
-
</p>
+
-
<p style="font-size: 1em;">
+
-
    &nbsp;
+
-
</p>
+
-
<p style="font-size: 1em;">
+
-
    &nbsp;
+
-
</p>
+
-
<p style="font-size: 1em;">
+
-
    &nbsp;
+
</p>
</p>
-
<table style="border: currentColor; border-image: none; width: 605px; margin-right: auto; margin-left: auto; border-collapse: collapse; styleName: Normal Table; border-insideH: 1px none #000000; border-insideV: 1px none #000000; cellpadding: 0px 7px 0px 7px;">
+
<table style="styleName:Table Grid;margin-left:0px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;border-insideH:1px solid #000000;border-insideV:1px solid #000000;cellpadding:0px 7px 0px 7px;styleName:Normal Table;margin-left:0px;cellpadding:0px 7px 0px 7px;width:605px;margin-left:auto;margin-right:auto;border-top:1px none #000000;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;border-insideH:1px none #000000;border-insideV:1px none #000000;border-collapse:collapse;border:none;font-family:times new roman;">
     <tbody>
     <tbody>
         <tr class="firstRow">
         <tr class="firstRow">
-
             <td style="padding: 0px 7px; border: 1px rgb(0, 0, 0); width: 605px; vertical-align: top;" colspan="2">
+
             <td style="width:605px;vertical-align:top;padding:0px 7px 0px 7px;border-top:1px none #000000;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;">
-
                 <p style="font-size: 1em;">
+
                 <p style="font-size:14px;">
-
                     <img width="589" height="340" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/3/35/RFU-T_924%2C999%2C716.png"/>
+
                     <img width="593.08" height="533.16" src="https://static.igem.org/mediawiki/2014/thumb/6/6a/Xmu-interlab-009.png/667px-Xmu-interlab-009.png" style="font-size:14px;"/><span style="font-size:14px;"> </span>
                 </p>
                 </p>
             </td>
             </td>
         </tr>
         </tr>
-
         <tr style="height: 227px;">
+
         <tr style="height:91px;">
-
             <td style="border-width: medium 1px 1px; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0); padding: 0px 7px; width: 605px; vertical-align: top;">
+
             <td style="width:605px;vertical-align:top;padding:0px 7px 0px 7px;border-top:none;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;">
-
                 <p style="font-size: 1em;">
+
                 <p style="font-size:14px;font-family:times new roman;">
-
                     <img width="291" height="226" style="font-size: 1em;background-color:beige;" src=" https://static.igem.org/mediawiki/parts/0/01/RFU-T_924.png"/>
+
                     <span style="font-weight:700;font-size:14px;"><strong>Figure</strong></span><span style="font-size:14px;"> </span><span style="font-weight:700;font-size:14px;"><strong>6.</strong></span><span style="font-size:14px;"> </span><span style="font-size:14px;">T</span><span style="font-size:14px;">he plot of RFUs versus time</span><span style="font-size:14px;">. Top row</span><span style="font-size:14px;"> are the plot of RFUs versus time</span><span style="font-size:14px;"> for each single device</span><span style="font-size:14px;">.</span><span style="font-size:14px;"> Bottom row are combination data of all three devices with error bar</span><span style="font-size:14px;">. F</span><span style="font-size:14px;">rom the plots</span><span style="font-size:14px;">, we can conclude that the RFUs increases linearly with time. </span><span style="font-size:14px;">C</span><span style="font-size:14px;">ompar</span><span style="font-size:14px;">ing</span><span style="font-size:14px;"> </span><span style="font-size:14px;">with</span><span style="font-size:14px;"> </span><span style="font-weight:700;font-size:14px;"><strong>Figure</strong></span><span style="font-size:14px;"> </span><span style="font-weight:700;font-size:14px;"><strong>5</strong></span><span style="font-size:14px;">, we can find that the GFP expression strength of</span><span style="font-size:14px;"> all</span><span style="font-size:14px;"> three devices from strong to weak is: <span>BBa_K1412924</span>, K1412716, K1412999. The relationship meets the assumption based on iGEM database. Because the </span><span style="font-size:14px;">activity of </span><span style="font-size:14px;">promoter J23101 is stronger than J23115, while the copies of backbone pSB1C3 is higher than pSB3K3. So </span><span style="font-size:14px;">that </span><span style="font-size:14px;">the result is reasonable.</span>
-
                </p>
+
-
            </td>
+
-
            <td style="border-width: medium 1px 1px medium; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0) currentColor; padding: 0px 7px; width: 605px; vertical-align: top;">
+
-
                <p style="font-size: 1em;font-family:Times New Roman;">
+
-
                    <span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">1</span><span style="font-size: 1em;"> is the combination of </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3</span><span style="font-size: 1em;"> and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;">. </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3</span><span style="font-size: 1em;"> and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;"> are the plots of RFUs versus time for each single device. From the plot of RFUs versus time, we can conclude that the RFUs </span><span style="font-size: 1em;">increases </span><span style="font-size: 1em;">linearly with time. By comparison in </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">1</span><span style="font-size: 1em;">, we can find that the GFP expression strength of three devices from strong to weak is: <span>BBa_K1412924</span>,<span>BBa_K1412716</span>,<span>BBa_K1412999</span>.</span><span style="font-size: 1em;"> </span><span style="font-size: 1em;">The relationship meets the assumption based on iGEM database. Because the promoter J23101 is stronger than J231</span><span style="font-size: 1em;">15, while the copies of backbone</span><span style="font-size: 1em;"> pSB1C3 is higher than pSB3K3. So the result is reasonable.</span>
+
                 </p>
                 </p>
             </td>
             </td>
         </tr>
         </tr>
-
         <tr style="height: 250px;">
+
         <tr style="height:0;">
-
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+
             <td style="width:605px;border:none;"></td>
-
                <p style="font-size: 1em;">
+
-
                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/3/34/RFU-T_999.png"/>
+
-
                </p>
+
-
            </td>
+
-
            <td style="border-width: medium 1px 1px medium; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0) currentColor; padding: 0px 7px; width: 605px; vertical-align: top;">
+
-
                <p style="font-size: 1em;">
+
-
                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/3/3f/RFU-T_716.png"/>
+
-
                </p>
+
-
            </td>
+
         </tr>
         </tr>
-
        <tr style="height: 0px;">
+
    </tbody>
-
             <td style="border: currentColor; border-image: none; width: 303px;"></td>
+
</table>
-
             <td style="border: currentColor; border-image: none; width: 302px;"></td>
+
<table style="styleName:Table Grid;margin-left:0px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;border-insideH:1px solid #000000;border-insideV:1px solid #000000;cellpadding:0px 7px 0px 7px;styleName:Normal Table;margin-left:0px;cellpadding:0px 7px 0px 7px;width:605px;margin-left:auto;margin-right:auto;border-top:1px none #000000;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;border-insideH:1px none #000000;border-insideV:1px none #000000;border-collapse:collapse;border:none;font-family:times new roman;">
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<table style="styleName:Table Grid;margin-left:0px;border-top:1px solid #000000;border-left:1px solid #000000;border-bottom:1px solid #000000;border-right:1px solid #000000;border-insideH:1px solid #000000;border-insideV:1px solid #000000;cellpadding:0px 7px 0px 7px;styleName:Normal Table;margin-left:0px;cellpadding:0px 7px 0px 7px;width:604px;margin-left:auto;margin-right:auto;border-top:1px none #000000;border-left:1px none #000000;border-bottom:1px none #000000;border-right:1px none #000000;border-insideH:1px none #000000;border-insideV:1px none #000000;border-collapse:collapse;border:none;font-family:times new roman;">
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                     <img width="589" height="340" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/7/7a/RFU-OD_924%2C999%2C716.png"/>
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                     <img width="599.15" height="494.31" src="https://static.igem.org/mediawiki/2014/6/65/Xmu-interlab-012.png" style="font-size:14px;"/>
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                 <p style="font-size:14px;font-family:Times New Roman;">
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                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/e/e3/RFU-OD_924.png"/>
+
                     <span style="font-weight:700;font-size:14px;"><strong>Figure</strong> </span><span style="font-weight:700;font-size:14px;"><strong>7.</strong></span><span style="font-size:14px;"> </span><span style="font-size:14px;">T</span><span style="font-size:14px;">he plot of RFUs versus OD</span><span style="font-size:14px;valign:sub;">600</span><span style="font-size:14px;valign:sub;">. </span><span style="font-size:14px;">Top row</span><span style="font-size:14px;"> are the plot of RFUs versus OD</span><span style="font-size:14px;valign:sub;">600</span><span style="font-size:14px;"> for each single device</span><span style="font-size:14px;">.</span><span style="font-size:14px;"> </span><span style="font-size:14px;"> </span><span style="font-size:14px;">Bottom row are combination data of all three devices with error bar</span><span style="font-size:14px;">.</span><span style="font-size:14px;"> </span><span style="font-size:14px;">From the plot</span><span style="font-size:14px;">,</span><span style="font-size:14px;"> we can find that the RFUs increases linearly with OD</span><span style="font-size:14px;valign:sub;">600</span><span style="font-size:14px;">, and we can conclude that </span><span style="font-size:14px;">the GFP expression strength of all three d</span><span style="font-size:14px;">evices from strong to weak is: </span><span>BBa_K1412924</span>, K1412716, K1412999.
-
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            <td style="border-width: medium 1px 1px medium; border-color: currentColor rgb(0, 0, 0) rgb(0, 0, 0) currentColor; padding: 0px 7px; width: 605px; vertical-align: top;">
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                 <p style="font-size: 1em;font-family:Times New Roman;">
+
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                     <span style="font-size: 1em; font-weight: 700;">Figure 1</span><span style="font-size: 1em;"> is the combination of </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3</span><span style="font-size: 1em;"> and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;">. </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3 </span><span style="font-size: 1em;">and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;"> are the plots of RFUs versus OD</span><span style="font-size: 1em; valign: sub;">600</span><span style="font-size: 1em;"> for each single device. From the plot of RFUs versus OD</span><span style="font-size: 1em; valign: sub;">600</span><span style="font-size: 1em;">, we can find that the RFUs </span><span style="font-size: 1em;">increases </span><span style="font-size: 1em;">linearly with OD</span><span style="font-size: 1em; valign: sub;">600</span><span style="font-size: 1em;">, and we can conclude that </span><span style="font-size: 1em;">the GFP expression strength of all three devices from strong to weak is: &nbsp;<span>BBa_K1412924</span>, <span>K1412716</span>,<span>K1412999</span>. </span><span style="font-size: 1em;">However, the linearity of </span><span style="font-size: 1em; font-weight: 700;">Figure </span><span style="font-size: 1em; font-weight: 700;">2</span><span style="font-size: 1em;"> is not very obvious. </span>
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                     <img width="589" height="340" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/5/5b/RFU_OD-T_924%2C999%2C716.png"/>
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                     <img width="588.96" height="517.48" src="https://static.igem.org/mediawiki/2014/d/d6/Xmu-interlab-013.png" style="font-size:14px;"/>
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                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/d/da/RFU_OD_T_924.png"/>
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                     <span style="font-weight:700;font-size:14px;"><strong>Figure</strong> </span><span style="font-weight:700;font-size:14px;"><strong>8.</strong></span><span style="font-size:14px;"> T</span><span style="font-size:14px;">he plots of RFUs/OD</span><span style="font-size:14px;valign:sub;">600</span><span style="font-size:14px;"> versus time for each single device. </span><span style="font-size:14px;">Top row</span><span style="font-size:14px;"> are the plot of RFUs/OD</span><span style="font-size:14px;valign:sub;">600</span><span style="font-size:14px;valign:sub;"> </span><span style="font-size:14px;">versus </span><span style="font-size:14px;">time for each single device</span><span style="font-size:14px;">.</span><span style="font-size:14px;"> </span><span style="font-size:14px;">From the plot of RFUs, we </span><span style="font-size:14px;">regard</span><span style="font-size:14px;valign:sub;"> </span><span style="font-size:14px;">the RFUs/OD</span><span style="font-size:14px;valign:sub;">600</span><span style="font-size:14px;"> as a</span><span style="font-size:14px;"> representation of the </span><span style="font-size:14px;">fluorescence </span><span style="font-size:14px;">expression </span><span style="font-size:14px;">strength</span><span style="font-size:14px;"> of unit bacteria. So we can get that the GFP expression strength of all three devices from strong to weak is: <span>BBa_K1412924</span>, <span>BBa_K1412716</span>, <span>BBa_K1412999</span>.</span>
-
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                 <p style="font-size: 1em;font-family:Times New Roman;">
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                     <span style="font-size: 1em; font-weight: 700;">Figure 1</span><span style="font-size: 1em;"> is the combination of </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3</span><span style="font-size: 1em;"> and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;">. </span><span style="font-size: 1em; font-weight: 700;">Figure</span><span style="font-size: 1em;"> </span><span style="font-size: 1em; font-weight: 700;">2, 3 </span><span style="font-size: 1em;">and </span><span style="font-size: 1em; font-weight: 700;">4</span><span style="font-size: 1em;"> are</span><span style="font-size: 1em;"> the plots of RFUs/OD</span><span style="font-size: 1em; valign: sub;">600</span><span style="font-size: 1em;"> versus time</span><span style="font-size: 1em;"> for each single device. From the plot of RFUs versus OD</span><span style="font-size: 1em; valign: sub;">600</span><span style="font-size: 1em;">, </span><span style="font-size: 1em;">we know the RFUs/OD</span><span style="font-size: 1em; valign: sub;">600</span><span style="font-size: 1em;"> is a representation of the fluoresent expression intensity of unit bacteria. So we can get that </span><span style="font-size: 1em;">the GFP expression strength of all three devices from strong to weak is: &nbsp;<span>BBa_K1412924</span>, <span>BBa_K1412716</span>, <span>BBa_K1412999</span>.</span>
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                    <img width="291" height="226" style="font-size: 1em;background-color:beige;" src="https://static.igem.org/mediawiki/parts/4/47/RFU_OD-T_716.png"/>
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    <span style="font-family: Wingdings;"></span><span style="font-family: Wingdings;"> </span><span style="font-size: 18px; font-weight: 700;">Reference:</span>
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     <span style="font-size: 1em;">[http://journals.aps.org/pre/abstract/10.1103/PhysRevE.82.021911 Bagh, Sangram, Mahuya Mandal, and David R. McMillen. &quot;Minimal genetic device with multiple tunable functions.&quot; Physical Review E 82.2 (2010): 021911].</span>
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     <span style="font-weight:700;font-size:18px;">Reference:</span>
</p>
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<p style="font-size: 2em; margin-left: 24px;">
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<p style="font-size: 14px;">
 +
    <span style="font-size:14px;"><a href="http://journals.aps.org/pre/abstract/10.1103/PhysRevE.82.021911" target="_self">1. Bagh, Sangram, Mahuya Mandal, and David R. McMillen. &quot;Minimal genetic device with multiple tunable functions.&quot; Physical Review E 82.2 (2010): 021911</a>.</span>
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<p style="font-size: 50px; margin-left: 24px;">
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Latest revision as of 03:50, 18 October 2014

side_bar


INTERLAB STUDY



The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. 

After the experiment, we get three parts:

BBa_K1412716: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 backbone.

BBa_K1412924: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), in the pSB1C3 backbone.

BBa_K1412999: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), in the pSB1C3 backbone.

 

GFP generator with J23101, J23115

For devices BBa_I20260 and BBa_J23101 + BBa_E0240. Both devices consist of Anderson promoter J23101 and GFP generator BBa_E0240. When the device is constructed in backbone pSB3K3. A low copy number is in expectation, as a result, a weak fluorescence strength is shown. While the device is constructed in pSB1C3 which is a higher copy number vector, hence a stronger fluorescence strength, so that it can be obvious enough to be observed in naked eyes.

For device BBa_J23115 + BBa_E0240, the promoter strength is the weakest, so that it’s difficult to observe green color in naked eyes.

 

Figure 1. GFP of different device under nature light. 1: BBa_K1412999 in DH5α; 2: BBa_K1412716 (reconstructed by us) in DH5α; 3: BBa_K1412716 in DH5α; 4: BBa_K1412924 in DH5α.

 

The bacteria was cultured in the LB medium for 12 hrs at 37 shaking at 200 rpm in the table concentrator, then 1 ml bacterium solution was transferred into 1.5 ml centrifuge tube, and was centrifuged at 10000 rcf(g) for 1 min. The supernatant was discarded and the residuals was suspend by PBS. The solution was centrifuged again, and we got the bacteria precipitate as the picture shown in Figure 1. In which we can find that device BBa_K1412924 is greenish in natural light while device BBa_K1412716 (reconstructed by us) and BBa_K1412716 emit a canary yellow color, and device BBa_K1412999 show the color which is close to white.

 

Figure 2. GFP of different devices under the UV-light. 1: BBa_K1412924 in DH5α; 2: BBa_K1412716 (reconstructed by us) in DH5α; 3: BBa_K1412716 in DH5α; 4: BBa_K1412999 in DH5α.

 

Under UV-light, the bacterium precipitate above can be observed clearly that device BBa_K1412924 can emit strong green fluorescence, while devices BBa_K1412716 (reconstructed by us) and BBa_K1412716 have weaker green fluorescence, and the green fluorescence from device BBa_K1412999 is the weakest so that we can’t even observe a green pixel.

 

 

Devices Verification




1.1kb Marker;

2.Double restriction enzyme digestion (EcoR I and Pst I) with device BBa_J23115 + BBa_E0240 (constructed by us).

3.Mono-restriction enzyme digestion (Pst I) with device BBa_J23115 + BBa_E0240 (constructed by XMU-China).

4.Double restriction enzyme digestion (EcoR I and Pst I) with device BBa_I20260 (reconstructed by us).

5.Mono-restriction enzyme digestion (Pst I) with device BBa_I20260 (reconstructed by us).

Figure 3. Enzyme digestion verification of devices BBa_J23115 + BBa_E0240 and BBa_I20260.

Results and discussion: For device BBa_I20260, it’s very abnormal that we get puzzling results with double enzymes digestion by Xba I and Pst I. The target segment seems vanished. However, if we use EcoR I and Pst I instead, we find that the segments, whose backbone is PSB3k3, generated by mono-restriction digestion is about 1000bp longer than that generated by double restriction enzyme digestion, and the devices have been verified by DNA sequencing. We can also get segments slightly shorter than 1000bp which generated by double digestion, and those segments are highlighted by red frames. So we confirm that device BBa_I20260 is correct. We took our actual measurement with the reconstructed device.








1.100 bp Marker.

2.BBa_K1412924 with double enzymes (Xba I and Pst I) digestion.

3.1kb Marker.


Figure 4. Enzyme digestion verification of devices BBa_J23101 + BBa_E0240

As double restriction enzyme digestion generates two target segments, we confirm that device BBa_J23101 + BBa_E0240 is constructed correctly.


Protocol

1. Transformed BBa_K1412924 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37.

2. Inoculate a 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate.

3. Cultures were grown in conical flask for 16 hrs at 37 with shaking at 200 rpm in the table concentrator.

4. Cultures were diluted 1:100 into three 20 ml fresh LB medium and grown for 3 hrs at 37 with shaking at 200 rpm in the table concentrator.

5. Then transfered 650 μl of the culture to a 1.5 ml centrifuge tube, centrifuged and washed twice with phosphate-buffered saline (PBS, pH 7.4) to minimize the background fluorescence from the medium.

6. The washed cells were suspended in PBS and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements.

7. Measure the fluorescence and absorbance:

(1)Fluorescence:

Device: SpectraMax+M5 microplate reader, 96-well plates.

Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.

(2)OD600 (optical density at 600 nm):

Device: SpectraMax+M5 microplate reader, 96-well plates.

Wavelengths: 600 nm absorption.

8. Measure every 30 minutes in the next 4 hrs.


Experimental data

Before the measurement, in order to set date in suitable range we estimated an appropriate concentration range by diluting the bacterium culture 2 times with PBS buffer. The values of PBS, the background, were subtracted during the data processing. And the data was removed when their deviations were too large. Then the remained data were doubled to get the final values to plot graph.

1.

Figure 5. The plot of OD versus time. Top row are the plots of OD versus time for each single device. Bottom row are combination data of all three devices with error bar. We can conclude that the growth rate is getting slower with time increasing. We measured each device with three samples for three times parallelly, and we make sure that the reproducibility of the data is acceptable. Comparing the plot of OD versus time with each other, we can see that their growth rate are almost the same.


Figure 6. The plot of RFUs versus time. Top row are the plot of RFUs versus time for each single device. Bottom row are combination data of all three devices with error bar. From the plots, we can conclude that the RFUs increases linearly with time. Comparing with Figure 5, we can find that the GFP expression strength of all three devices from strong to weak is: BBa_K1412924, K1412716, K1412999. The relationship meets the assumption based on iGEM database. Because the activity of promoter J23101 is stronger than J23115, while the copies of backbone pSB1C3 is higher than pSB3K3. So that the result is reasonable.


Figure 7. The plot of RFUs versus OD600. Top row are the plot of RFUs versus OD600 for each single device. Bottom row are combination data of all three devices with error bar. From the plot, we can find that the RFUs increases linearly with OD600, and we can conclude that the GFP expression strength of all three devices from strong to weak is: BBa_K1412924, K1412716, K1412999.


Figure 8. The plots of RFUs/OD600 versus time for each single device. Top row are the plot of RFUs/OD600 versus time for each single device. From the plot of RFUs, we regard the RFUs/OD600 as a representation of the fluorescence expression strength of unit bacteria. So we can get that the GFP expression strength of all three devices from strong to weak is: BBa_K1412924, BBa_K1412716, BBa_K1412999.


Reference:

1. Bagh, Sangram, Mahuya Mandal, and David R. McMillen. "Minimal genetic device with multiple tunable functions." Physical Review E 82.2 (2010): 021911.