Team:Paris Bettencourt/Project/Interlab Study

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<body>
<body>
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<h4><big>iGEM 2014 Measurement Interlab Study</big></h4>
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<p id=top><a href="#topheader"><img src="https://static.igem.org/mediawiki/2014/d/d3/Arrowpb.png"></a></p>
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<div id="topheader">
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<p>iGEM Paris Bettencourt team participates in the <a href="https://2014.igem.org/Tracks/Measurement/Interlab_study iGEM interlab study">Interlab study</a></p>
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<img src="https://static.igem.org/mediawiki/2014/b/b1/Smelltheroses.png" class=nameimg>
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</div>
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<p><I>"The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. Can you measure fluorescence somewhere in your lab? Then this is the perfect study for you! Even if your lab or the organisms you work with mean that you can’t measure GFP from the specific devices, we want every team to be able to participate: email measurement at igem dot org and we will work out an alternative."</I></p>
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<div id=logo><img id=logoblanc src="https://static.igem.org/mediawiki/2014/7/7d/Logoblanc.png"><img id=logorose src="https://static.igem.org/mediawiki/2014/2/28/Logorose.png"></div>
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<div id=fond>
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<img id=illustration src="https://static.igem.org/mediawiki/2014/3/35/GFP.jpg"|center>
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<p><b>Introduction</b></br>
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<i>"The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. Can you measure fluorescence somewhere in your lab? Then this is the perfect study for you! Even if your lab or the organisms you work with mean that you can’t measure GFP from the specific devices, we want every team to be able to participate: email measurement at igem dot org and we will work out an alternative."</i></p>
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</div>
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<div id=separation></div>
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<table id=tablelien>
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<div>
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<tr>
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<h6>First device</h6>
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<td><a href="#devices">Devices</a></td>
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<div id=image2><img id=image1 src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p id=definition>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
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<td><a href="#results">Results</a></td>
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<p><u>BBa_I20260</u> (J23101-B0032-E0040-B0010-B0012) in the <u>pSB3K3</u> vector.</p>
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<td><a href="#"></a></td>
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<p>Selection marker : Kanamycin</p>
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<td><a href="#motivation">Motivations</a></td>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</p>
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</tr>
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</table>
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<ul><u>2012 BioBrick Kit location</u>
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<div id=devices>
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  <li> BBa_I20260: Plate 2, Well 17F</li>
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<h5>Devices</h5>
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</ul>
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<div class=image><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
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</div>
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<h6>Device 1</h6>
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<div class=text><p><u>BBa_I20260</u> (J23101-B0032-E0040-B0010-B0012) in the <u>pSB3K3</u> vector.</p>
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<p>Selection marker : Kanamycin</p>
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<div><p> I followed <a href=kit>iGEM Distribution Kit instructions </a> to extract DNA from the Biobrick Plate 2, Well 17F (2012) and then <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1"> Heat Shock transformation of <i>E.coli</i></a></br>For successful Kanymycin plates I prepared <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a> and labbeled it G.22.</p>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</p>
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</div>
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<u>2012 BioBrick Kit location</u>
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<div id=separation></div>
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  <ul><li>BBa_I20260: Plate 2, Well 17F</li>
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<div>
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</ul>
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<h6>Second device</h6>
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<p> I followed <a href=kit>iGEM Distribution Kit instructions </a> to extract DNA from the Biobrick Plate 2, Well 17F (2012) and then <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1"> Heat Shock transformation of <i>E.coli</i></a></br>For successful Kanymycin plates I prepared <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a> and labbeled it G.22.</p>
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<div id=image2><img id=image1 src="https://static.igem.org/mediawiki/2014/0/02/Device_2.png"><p id=definition>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
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</div>
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<p><u>BBa_J23101 + BBa_E0240</u> (B0032-E0040-B0010-B0012),  in the <u>pSB1C3</u> vector.</p>
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<div class=image><img src="https://static.igem.org/mediawiki/2014/0/02/Device_2.png"><p class=definition>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
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<p>Selection marker : Chloramphenicol</p>
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<h6>Device 2</h6>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</p>
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<div class=text>
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<p><u>BBa_J23101 + BBa_E0240</u> (B0032-E0040-B0010-B0012),  in the <u>pSB1C3</u> vector.</br>
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<ul><u>2014 Biobrick Kit locations</u>
+
Selection marker : Chloramphenicol</br>
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  <li>  BBa_K823005 (BBa_J23101 in pSB1C3): Plate 1, Well 20K</li>
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Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</p>
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  <li>  BBa_E0240 (in pSB1C3): Plate 2, Well 24B</li>
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<u>2014 Biobrick Kit locations</u>
-
</ul>
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  <ul><li>  BBa_K823005 (BBa_J23101 in pSB1C3): Plate 1, Well 20K</li>
-
</div>
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  <li>  BBa_E0240 (in pSB1C3): Plate 2, Well 24B</li>
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+
</ul>
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<div><p> I followed <a href=kit>iGEM Distribution Kit instructions </a> to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1"> Heat Shock transformation of <i>E.coli</i></a></br>For successful Chloramphenicol plates,  form single colonies I prepared liquid cultures overnight. I used 750uL of the liquid cultures for a<a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a>. I used remaining 4,25 mL to make <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4"> minipreps  </a>. I measured DNA content with the nanodrop.</br><u>Digestion analysis</u>:</br>
+
<p> I followed <a>iGEM Distribution Kit instructions </a> to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1"> Heat Shock transformation of <i>E.coli</i></a></br>For successful Chloramphenicol plates,  form single colonies I prepared liquid cultures overnight. I used 750uL of the liquid cultures for a<a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a>. I used remaining 4,25 mL to make <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4"> minipreps  </a>. I measured DNA content with the nanodrop.</br></br><u>Digestion analysis</u>:</br>
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- 5 ug plasmid </br>
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<ul><li>- 5 ug plasmid </li>
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- 5 ul FD Buffer</br>
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<li>- 5 ul FD Buffer</li>
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- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)</br>
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<li>- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)</li>
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- complete with H2O</br>
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<li>- complete with H2O</li></ul>
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(Final volume of 50 uL)</br>
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(Final volume of 50 uL)</br>
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</br> We made an eletrophoresis gel to check the fragments (the bands at around  876 bp for GFP and 2100 bp for the promoter + backbone)and then extract BBa_E0240 with Gel extraction kit. For the plasmid with the promoter I used a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot5">PCR purification kit.</a>
+
We made an eletrophoresis gel to check the fragments (the bands at around  876 bp for GFP and 2100 bp for the promoter + backbone)and then extract BBa_E0240 with Gel extraction kit. For the plasmid with the promoter I used a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot5">PCR purification kit.</a>
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</br> I introduced the GFP fragment to the Plasmid + backbone through ligation of the sticky ends SpeI and XbeI. Quantified DNA in two parts with nanodrop. The amount of  vector:insert has been calculated with Promega calculator.  
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I introduced the GFP fragment to the Plasmid + backbone through ligation of the sticky ends SpeI and XbeI. Quantified DNA in two parts with nanodrop. The amount of  vector:insert has been calculated with Promega calculator.  
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</br>
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</br>
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</br>5X Ligase Reaction Buffer 4 μl
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<ul><li>5X Ligase Reaction Buffer 4 μl</li>
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</br>Insert: Vector Molar Ratio 1:1, 1:3, 1:5
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<li>Insert: Vector Molar Ratio 1:1, 1:3, 1:5</li>
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</br>Total DNA 0.01-0.1 μg
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<li>Total DNA 0.01-0.1 μg</li>
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</br>T4 DNA Ligase 1 uL
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<li>T4 DNA Ligase 1 uL</li>
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</br>Autoclaved distilled water to 25uL
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<li>Autoclaved distilled water to 25uL</li>
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</br>
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<li>Incubate at 22°C for 1h</li>
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</br>Incubate at 22°C for 1h
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<li> 16°C overnight</li></ul>
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</br> 16°C overnight
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I transformed the ligation product following <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1"> Heat Shock transformation of <i>E.coli</i></a>.
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</br>
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I have put a single colony into a liquid culture with the appropriate antibiotic and the next day I prepared a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a>.</p>
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</br> I transformed the ligation product following <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1"> Heat Shock transformation of <i>E.coli</i></a>.
+
</div>
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</br>I have put a single colony into a liquid culture with the appropriate antibiotic and the next day I prepared a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a>.
+
<div class=image><img src="https://static.igem.org/mediawiki/2014/d/d9/Device_3.png"><p class=definition>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
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</div></p>
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<h6>Device 3</h6>
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<div id=separation></div>
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<div class=text>
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<div>
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<p><i>*BBa_J23115 was cloned using BBa_K823012 and therefore should have 2 missmatched basepairs.</i></p>
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<h6>Third device*</h6>
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<p><u>BBa_J23115 + BBa_E0240</u> (B0032-E0040-B0010-B0012),  in the <u>pSB1C3</u> vector.</br>
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<div id=image2><img id=image1 src="https://static.igem.org/mediawiki/2014/d/d9/Device_3.png"><p id=definition>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</br></p></div>
+
Selection marker : Chloramphenicol</br>
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<p><i>*BBa_J23115 was cloned using BBa_K823012 and therefore should have 2 missmatched basepairs.</i></p>
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Promoter expected sequence : tttatagctagctcag<u>t</u>cct<u>a</u>ggtacaatgctagc (missmatched basepairs compared to real BBa_J23115 are underlined)</p>
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<p><u>BBa_J23115 + BBa_E0240</u> (B0032-E0040-B0010-B0012),  in the <u>pSB1C3</u> vector.</p>
+
<u>2014 Biobrick Kit locations</u>
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<p>Selection marker : Chloramphenicol</p>
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   <ul><li>  BBa_K823012 (BBa_J23115 in pSB1C3): Plate 1, Well 22I</li>
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<p>Promoter expected sequence : tttatagctagctcag<u>t</u>cct<u>a</u>ggtacaatgctagc (missmatched basepairs compared to real BBa_J23115 are underlined)</p>
+
-
 
+
-
<ul><u>2014 Biobrick Kit locations</u>
+
-
   <li>  BBa_K823012 (BBa_J23115 in pSB1C3): Plate 1, Well 22I</li>
+
   <li>  BBa_E0240 (in pSB1C3): Plate 2, Well 24B </li>
   <li>  BBa_E0240 (in pSB1C3): Plate 2, Well 24B </li>
</ul>
</ul>
</br>
</br>
<p>In order to prepare the third device we proceed exactly in the same way as for the Device 2, except we used  BBa_K823012 instead of BBa_K823005</p>
<p>In order to prepare the third device we proceed exactly in the same way as for the Device 2, except we used  BBa_K823012 instead of BBa_K823005</p>
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<h3>Results</h3>
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</div>
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</br>
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</div>
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</br>
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<div id=results>
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<h6><u>Sequencing</u></h6>
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<h5>Sequencing</h5>
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<p> We sequenced all the three devices using iGEM Verification Primers (forward). Sequences are matching expected constructs.</p>
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<p>We sequenced all the three devices using iGEM Verification Primers (forward). Sequences are matching expected constructs.</p>
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</br>
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<div><img src="https://static.igem.org/mediawiki/2014/0/0a/OD600_min.jpg"><p>Mean OD600 absorbance measured over 20h. Background absorbance (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for each sample).</p></div>
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<h6>First device</h6>
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<h6>First Device</h6>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</p>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</br>
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<p>Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC</p>
+
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC</br>
-
<p>Complete sequenced device:</br>
+
Complete sequenced device:</br>
-
ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA</p>
+
ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA</p>
-
<h6>Second device</h6>
+
<div><img src="https://static.igem.org/mediawiki/2014/e/e7/Fluo_samples_min.jpg"><p>Mean of green fluorescence for three devices and NEB turbo cells. </br>Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).</p></div>
-
<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</p>
+
<h6>Second Device</h6>
-
<p>Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC</p>
+
<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</br>
-
<p>Complete sequenced device:</br>
+
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC</br>
-
TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA
+
Complete sequenced device:</br>
-
</p>
+
TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA</p>
-
<h6>Third device</h6>
+
<div><img src="https://static.igem.org/mediawiki/2014/d/d0/Fluo_od_min.jpg"><p>Mean of green fluorescence divided by optical density 600 for three devices and NEB turbo cells. </br>Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).</p></div>
-
<p>Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc </p>
+
<h6>Third Device</h6>
-
<p>Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC</p>
+
<p>Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc</br>
-
<p>Complete sequence: </br>
+
Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC</br>
-
GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA
+
Complete sequence: </br>
-
</p>
+
GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA</p>
-
</br>
+
<div><img src="https://static.igem.org/mediawiki/2014/3/35/GFP.jpg"></div>
-
</br>
+
<h5>OD600 and fluorescence measure over 20h</h5>
-
<h6><u>OD600 and fluorescence measure over 20h</u></h6>
+
<p><u>Samples preparation:</u> Single colonies were inoculated in 5mL LB broth with appropriate antibiotic and grown to saturation overnight (16h) at 37°C with shaking (220 rpm).
-
<p><u>Samples preparation:</u> Single colonies were inoculated in 5mL LB broth with appropriate antibiotic and grown to saturation overnight (16h) at 37°C with shaking (220 rpm).
+
Samples were diluted 100x (50um in 5 mL LB with appropriate antibiotic) and incubated for 2h at 37°C prior to measurement.
-
</br>
+
</br></br><u>Control:</u></br>
-
Samples were diluted 100x (50um in 5 mL LB with appropriate antibiotic) and incubated for 2h at 37°C prior to measurement.
+
LB broth with antibiotics (chloramphenicol/kanamycin)- no fluorescence</br>
-
</p>
+
NEB turbo without fluorescence - no fluorescence, no cells</br>
-
<p><ul><u>Control:</u> <li>LB broth with antibiotics (chloramphenicol/kanamycin)- no fluorescence</li><li> NEB turbo without fluorescence - no fluorescence, no cells</li></ul>
+
<u>Measurment</u></br>
-
<p>
+
Greiner 96 plates were loaded with 150um of cells in LB and 30um mineral oil
-
<p>
+
Cells have been diluted prior to measurement as described above.
-
<u>Measurment</u>
+
Background absorbance and fluorescence was determined from LB control.
-
Greiner 96 plates were loaded with 150um of cells in LB and 30um mineral oil
+
</br>Data from the top row were excluded due to the likely evaporation and artefacts (edge effects).  
-
Cells have been diluted prior to measurement as described above.
+
</p>
-
Background absorbance and fluorescence was determined from LB control.
+
</div>
-
</br>Data from the top row were excluded due to the likely evaporation and artefacts (edge effects).  
+
-
</p><p>
+
-
 
+
-
<div id=illust2 ><img id=illustration2 src="https://static.igem.org/mediawiki/2014/0/0a/OD600_min.jpg"><p>Mean OD600 absorbance measured over 20h. Background absorbance (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for each sample).</p></div>
+
-
 
+
-
<div id=illust2><img id=illustration2 src="https://static.igem.org/mediawiki/2014/e/e7/Fluo_samples_min.jpg"><p>Mean of green fluorescence for three devices and NEB turbo cells. </br>Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).</p></div>
+
-
 
+
-
<div id=illust2><img id=illustration2 src="https://static.igem.org/mediawiki/2014/d/d0/Fluo_od_min.jpg"><p>Mean of green fluorescence divided by optical density 600 for three devices and NEB turbo cells. </br>Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).</p></div>
+
-
 
+
-
<div id=separation></div>
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</body>
</body>
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Revision as of 16:00, 1 October 2014

Introduction
"The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. Can you measure fluorescence somewhere in your lab? Then this is the perfect study for you! Even if your lab or the organisms you work with mean that you can’t measure GFP from the specific devices, we want every team to be able to participate: email measurement at igem dot org and we will work out an alternative."

Devices Results Motivations
Devices

Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​

Device 1

BBa_I20260 (J23101-B0032-E0040-B0010-B0012) in the pSB3K3 vector.

Selection marker : Kanamycin

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc

2012 BioBrick Kit location
  • BBa_I20260: Plate 2, Well 17F

I followed iGEM Distribution Kit instructions to extract DNA from the Biobrick Plate 2, Well 17F (2012) and then Heat Shock transformation of E.coli
For successful Kanymycin plates I prepared glycerol stock and labbeled it G.22.

Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​

Device 2

BBa_J23101 + BBa_E0240 (B0032-E0040-B0010-B0012), in the pSB1C3 vector.
Selection marker : Chloramphenicol
Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc

2014 Biobrick Kit locations
  • BBa_K823005 (BBa_J23101 in pSB1C3): Plate 1, Well 20K
  • BBa_E0240 (in pSB1C3): Plate 2, Well 24B

I followed iGEM Distribution Kit instructions to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then Heat Shock transformation of E.coli
For successful Chloramphenicol plates, form single colonies I prepared liquid cultures overnight. I used 750uL of the liquid cultures for aglycerol stock . I used remaining 4,25 mL to make minipreps . I measured DNA content with the nanodrop.

Digestion analysis:

  • - 5 ug plasmid
  • - 5 ul FD Buffer
  • - 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
  • - complete with H2O
(Final volume of 50 uL)
We made an eletrophoresis gel to check the fragments (the bands at around 876 bp for GFP and 2100 bp for the promoter + backbone)and then extract BBa_E0240 with Gel extraction kit. For the plasmid with the promoter I used a PCR purification kit. I introduced the GFP fragment to the Plasmid + backbone through ligation of the sticky ends SpeI and XbeI. Quantified DNA in two parts with nanodrop. The amount of vector:insert has been calculated with Promega calculator.
  • 5X Ligase Reaction Buffer 4 μl
  • Insert: Vector Molar Ratio 1:1, 1:3, 1:5
  • Total DNA 0.01-0.1 μg
  • T4 DNA Ligase 1 uL
  • Autoclaved distilled water to 25uL
  • Incubate at 22°C for 1h
  • 16°C overnight
I transformed the ligation product following Heat Shock transformation of E.coli. I have put a single colony into a liquid culture with the appropriate antibiotic and the next day I prepared a glycerol stock .

Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​

Device 3

*BBa_J23115 was cloned using BBa_K823012 and therefore should have 2 missmatched basepairs.

BBa_J23115 + BBa_E0240 (B0032-E0040-B0010-B0012), in the pSB1C3 vector.
Selection marker : Chloramphenicol
Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc (missmatched basepairs compared to real BBa_J23115 are underlined)

2014 Biobrick Kit locations
  • BBa_K823012 (BBa_J23115 in pSB1C3): Plate 1, Well 22I
  • BBa_E0240 (in pSB1C3): Plate 2, Well 24B

In order to prepare the third device we proceed exactly in the same way as for the Device 2, except we used BBa_K823012 instead of BBa_K823005

Sequencing

We sequenced all the three devices using iGEM Verification Primers (forward). Sequences are matching expected constructs.

Mean OD600 absorbance measured over 20h. Background absorbance (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for each sample).

First Device

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC
Complete sequenced device:
ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA

Mean of green fluorescence for three devices and NEB turbo cells.
Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).

Second Device

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC
Complete sequenced device:
TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA

Mean of green fluorescence divided by optical density 600 for three devices and NEB turbo cells.
Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).

Third Device

Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc
Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC
Complete sequence:
GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA

OD600 and fluorescence measure over 20h

Samples preparation: Single colonies were inoculated in 5mL LB broth with appropriate antibiotic and grown to saturation overnight (16h) at 37°C with shaking (220 rpm). Samples were diluted 100x (50um in 5 mL LB with appropriate antibiotic) and incubated for 2h at 37°C prior to measurement.

Control:
LB broth with antibiotics (chloramphenicol/kanamycin)- no fluorescence
NEB turbo without fluorescence - no fluorescence, no cells
Measurment
Greiner 96 plates were loaded with 150um of cells in LB and 30um mineral oil Cells have been diluted prior to measurement as described above. Background absorbance and fluorescence was determined from LB control.
Data from the top row were excluded due to the likely evaporation and artefacts (edge effects).

Retrieved from "http://2014.igem.org/Team:Paris_Bettencourt/Project/Interlab_Study"