Team:Paris Bettencourt/Project/Interlab Study

From 2014.igem.org

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<div id=fond>
<div id=fond>
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<table id=headtable>
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<tr>
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<td><b>BACKGROUND</b></br><br>
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The first international interlab measurement study in synthetic biology!</td>
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<td><b>AIMS</b></br><br>
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Measure GFP expression under different Anderson promoters and in different plasmids.</td>
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<td><b>RESULTS</b></br><br>
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<ul>
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<li>Successfully built the three devices</li>
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<li>Sequenced and gel verified the three devices</li>
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<li>Characterised all the three devices with the OD600 and fluorescence measurements in a Tecan micro-plate reader</li><li>- Reported the results on the <a href="https://2014.igem.org/Team:Paris_Bettencourt/Project/Interlab_Study">Interlab Study</a> page of the iGEM wiki</li>
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<li>Completed the description of the <a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> - Device 1
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<li>Submitted and send parts: <a href="http://parts.igem.org/Part:BBa_K1403000">BBa_K1403000</a> - Device 2 and <a href="http://parts.igem.org/Part:BBa_K1403001">BBa_K1403001</a> - Device 3 to the Biobricks registry</li> </ul></td>
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         </div>
         </div>
<table id=tablelien>
<table id=tablelien>
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<div class=division>
<div class=division>
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<h5>Achievements</h5>
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<p class=text2><img id=imgfluo src="https://static.igem.org/mediawiki/2014/b/bd/GFPpb.jpg"></br><span class=definition><b>Photo 1. Fluorescent devices and LB control.</b> A photo of the three devices and an LB control was taken with a help of transillumiantor</span></p>
 +
<p class=text1><ul>
 +
<li>Successfully built the three devices</li>
 +
<li>Sequenced and gel verified the three devices</li>
 +
<li>Characterised all the three devices with the OD600 and fluorescence measurements in a Tecan micro-plate reader</li><li>Reported the results on the <a href="https://2014.igem.org/Team:Paris_Bettencourt/Project/Interlab_Study">Interlab Study</a> page of the iGEM wiki</li>
 +
<li>Completed the description of the <a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Device 1)
 +
<li>Submitted and send parts: <a href="http://parts.igem.org/Part:BBa_K1403000">BBa_K1403000</a> (Device 2) and <a href="http://parts.igem.org/Part:BBa_K1403001">BBa_K1403001</a> (Device 3) to the Biobricks registry</li> </ul></p></div>
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<div class=division>
</br><h5>Introduction</h5>
</br><h5>Introduction</h5>
<p class=text1><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. "</i></p>
<p class=text1><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. "</i></p>
<h5>Motivation</h5>
<h5>Motivation</h5>
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<p class=text1>iGEM Paris Bettencourt team supported this initiative with pleasure. We strongly believe that this study will help to define standards for BioBrick constructs and measure the difference between different labs and measurement methods. It was also a great training for inexperienced synthetic biologist of the team which allowed as to train transformation and ligation techniques. </p>
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<p class=text1>iGEM Paris Bettencourt team supported this initiative with pleasure. We strongly believe that this study will help to define standards for BioBrick constructs and measure the difference between different labs and measurement methods. It was also a great training for transformation and ligation techniques for the inexperienced synthetic biologist on the team.</p>
</div>
</div>
</br></br></br><h5>Devices</h5>
</br></br></br><h5>Devices</h5>
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<div class=text><p>We build 3 devices according to <a href"https://2014.igem.org/Tracks/Measurement/Interlab_study">Interlab study instructions</a>. First designed in a software (Geneious v. 7.0.6). Then realised in our wet lab out of Biobrick Distribution Kits. Confirmed by eletrophoresis gel analytic dgestion as well as sequencing. Finally, stocked as a glycerol stock and used for the measurments of GFP expression. </p></div>
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<div class=text><p>We built 3 devices (Fig. 1, Fig. 2, Fig. 3) according to <a href"https://2014.igem.org/Tracks/Measurement/Interlab_study">Interlab study instructions</a>. First designed in software (Geneious v. 7.0.6). Then made in our wet lab using the Biobrick Distribution Kits and confirmed by electrophoresis gel analytic digestion as well as sequencing. Finally, stocked as a glycerol stock and used for the measurements of GFP expression. </p></div>
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<div class=image id=firstimg><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 class=image id=firstimg><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition><b>Figure 1. Device 1.</b> Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
</br><h6>Device 1</h6></br>
</br><h6>Device 1</h6></br>
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<div class=image id=firstimg><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 class=image id=firstimg><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition><b>Figure 1. Device 1.</b> Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
<div class=text><p><u>BBa_I20260</u> (J23101-B0032-E0040-B0010-B0012) in the <u>pSB3K3</u> vector.</p>
<div class=text><p><u>BBa_I20260</u> (J23101-B0032-E0040-B0010-B0012) in the <u>pSB3K3</u> vector.</p>
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  <ul><li>BBa_I20260: Plate 2, Well 17F</li>
  <ul><li>BBa_I20260: Plate 2, Well 17F</li>
</ul>
</ul>
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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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<p> We 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 transformed the <i>E. coli</i></a> colonies that grew in the selective Kanymycin were grown in liquid media and made into <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stocks</a> labelled G.22.</p>
</div>
</div>
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                 <div id=divimage class=image><div class=separation2></div><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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                 <div id=divimage class=image><div class=separation2></div><img src="https://static.igem.org/mediawiki/2014/0/02/Device_2.png"><p class=definition><b>Figure 2. Device 2.</b>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
</br></br><h6>Device 2</h6></br>
</br></br><h6>Device 2</h6></br>
<div class=text>
<div class=text>
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  <li>  BBa_E0240 (in pSB1C3): Plate 2, Well 24B</li>
  <li>  BBa_E0240 (in pSB1C3): Plate 2, Well 24B</li>
</ul>
</ul>
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<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.
+
<p> We followed the <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 transformed <i>E. coli</i></a></br>The colonies that survived after selection in choloamphenicol were cultured overnight. We used 750uL of the liquid cultures for a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock </a>. The remaining 4.25 mL were used to make <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4"> minipreps </a>. We measured DNA content with the nanodrop.
</br></br><u>Digestion analysis</u>:</br>
</br></br><u>Digestion analysis</u>:</br>
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<ul><li>- 5 ug plasmid </li>
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<ul><li> 5 ug plasmid </li>
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<li>- 5 ul FD Buffer</li>
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<li> 5 ul Buffer</li>
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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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<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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<li>- complete with H2O</li></ul>
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<li> Complete with H2O</li></ul>
(Final volume of 50 uL)</br>
(Final volume of 50 uL)</br>
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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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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 extracted BBa_E0240 with a gel extraction kit. For the plasmid with the promoter we used a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot5">PCR purification kit.</a>
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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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We 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.  
</br>
</br>
<ul><li>5X Ligase Reaction Buffer 4 μl</li>
<ul><li>5X Ligase Reaction Buffer 4 μl</li>
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<li>Incubate at 22°C for 1h</li>
<li>Incubate at 22°C for 1h</li>
<li> 16°C overnight</li></ul>
<li> 16°C overnight</li></ul>
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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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We 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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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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We made liquid cultures of single colonies with the appropriate antibiotic and the next day we prepared a <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8">glycerol stock</a>.</p>
</div>
</div>
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<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 class=image><img src="https://static.igem.org/mediawiki/2014/d/d9/Device_3.png"><p class=definition><b>Figure 3. Device 3.</b>Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​</p></div>
</br></br><h6>Device 3</h6></br>
</br></br><h6>Device 3</h6></br>
<div class=text>
<div class=text>
<p>Our part in the registry: <a href="http://parts.igem.org/Part:BBa_K1403001">BBa_K1403001</a></p>
<p>Our part in the registry: <a href="http://parts.igem.org/Part:BBa_K1403001">BBa_K1403001</a></p>
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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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<p><i>*BBa_J23115 was cloned using BBa_K823012 and therefore should have 2 mismatched basepairs.</i></p>
<p><u>BBa_J23115 + BBa_E0240</u> (B0032-E0040-B0010-B0012),  in the <u>pSB1C3</u> vector.</br>
<p><u>BBa_J23115 + BBa_E0240</u> (B0032-E0040-B0010-B0012),  in the <u>pSB1C3</u> vector.</br>
Selection marker : Chloramphenicol</br>
Selection marker : Chloramphenicol</br>
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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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Promoter expected sequence : tttatagctagctcag<u>t</u>cct<u>a</u>ggtacaatgctagc (mismatched basepairs compared to real BBa_J23115 are underlined)</p>
<u>2014 Biobrick Kit locations</u>
<u>2014 Biobrick Kit locations</u>
   <ul><li>  BBa_K823012 (BBa_J23115 in pSB1C3): Plate 1, Well 22I</li>
   <ul><li>  BBa_K823012 (BBa_J23115 in pSB1C3): Plate 1, Well 22I</li>
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</ul>
</ul>
</br>
</br>
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<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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<p>In order to prepare the third device we proceeded exactly in the same way as for the Device 2, except we used  BBa_K823012 instead of BBa_K823005</p>
</div>
</div>
</div>
</div>
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</br></br></br><h5>Sequencing</h5>
</br></br></br><h5>Sequencing</h5>
<p>We sequenced all the three devices using iGEM Verification Primers (forward). Sequences are matching expected constructs.</p>
<p>We sequenced all the three devices using iGEM Verification Primers (forward). Sequences are matching expected constructs.</p>
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</br></br><h6>First Device</h6></br>
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</br><h6>1. Device 1</h6>
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<div>...GAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA</div>
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<div></br></br></br></br>...GTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA</div>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</br>
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<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</br></br>
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Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC</br>
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Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC</br></br>
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Complete sequenced device:</br>
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Complete sequenced device:</br></br>
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ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATAT...</p>
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ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAA...</p>
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</br></br><h6>Second Device</h6></br>
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</br></br><h6>2. Device 2</h6>
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<div>...AGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA</div>
+
<div></br></br></br></br>...AGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA</div>
-
<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</br>
+
<p>Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc</br></br>
-
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC</br>
+
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC</br></br>
-
Complete sequenced device:</br>
+
Complete sequenced device:</br></br>
TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGA...</p>
TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGA...</p>
-
</br></br><h6>Third Device</h6></br>
+
</br></br><h6>3. Device 3</h6>
-
<div>...AAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA</div>
+
<div></br></br></br></br>...GTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA</div>
-
<p>Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc</br>
+
<p>Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc</br></br>
-
Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC</br>
+
Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC</br></br>
-
Complete sequence: </br>
+
Complete sequence: </br></br>
-
GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAA...</p>
+
GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAA...</p>
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<div><img src="https://static.igem.org/mediawiki/2014/3/35/GFP.jpg"></div>
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<div><img src="https://static.igem.org/mediawiki/2014/0/0a/OD600_min.jpg"></br><b><span class=definition>Figure 4. Mean OD600 absorbance measured over 20h.</b> Background absorbance (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for each sample).</span></br><img src="https://static.igem.org/mediawiki/2014/e/e7/Fluo_samples_min.jpg"></br><span class=definition><b>Figure 5. Mean of green fluorescence for three devices and NEB turbo cells.</b> </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).</span></br>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d0/Fluo_od_min.jpg"></br><span class=definition><b>Figure 6. Mean of green fluorescence divided by optical density 600 for three devices and NEB turbo cells.</b> </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).</span></div>
</br></br></br><h5>OD600 and fluorescence measure over 20h</h5>
</br></br></br><h5>OD600 and fluorescence measure over 20h</h5>
<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).
Line 364: Line 356:
</p>
</p>
</br></br></br><h5>Results</h5>
</br></br></br><h5>Results</h5>
-
<p>Here we present the result of the measurments. The measument has been realised in a comparable growth conditions for all Divices (Fig.1). We can clearly see that the highest GFP expression levels occures under <a href="http://parts.igem.org/Part:BBa_J23101"> BBa_J23101</a> Anderson's strong promoter in a high copy plasmid sPB1C3 (Device 2: <a href="http://parts.igem.org/Part:BBa_K1403000">BBa_K1403000</a>). The lowest GFP levels occures under very weak Anderson's promoter - mutated <a href="http://parts.igem.org/Part:BBa_J23101"> J23115</a> (Device 3: <a href="http://parts.igem.org/Part:BBa_K1403001">BBa_K1403001</a>) </p>
+
<p>Here we present the result of the measurments. The measument has been realised in a comparable growth conditions for all Devices (Fig. 4). We can clearly see that the highest GFP expression levels occures under <a href="http://parts.igem.org/Part:BBa_J23101"> BBa_J23101</a> Anderson's strong promoter in a high copy plasmid sPB1C3 (Device 2: <a href="http://parts.igem.org/Part:BBa_K1403000">BBa_K1403000</a>). The lowest GFP levels occures under very weak Anderson's promoter - mutated <a href="http://parts.igem.org/Part:BBa_J23101"> J23115</a> (Device 3: <a href="http://parts.igem.org/Part:BBa_K1403001">BBa_K1403001</a>) as can be seen in the Fig. 5 & Fig. 6 </p>
</div>
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<p class=text1><img src="https://static.igem.org/mediawiki/2014/0/0a/OD600_min.jpg"></br>Fig.1 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>
+
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<p class=text1><img src="https://static.igem.org/mediawiki/2014/e/e7/Fluo_samples_min.jpg"></br>Fig.2 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>
+
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<p class=text1><img src="https://static.igem.org/mediawiki/2014/d/d0/Fluo_od_min.jpg"></br>Fig.3 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>
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{{:Team:Paris_Bettencourt/Footer}}
{{:Team:Paris_Bettencourt/Footer}}

Latest revision as of 16:05, 7 December 2014

Devices Sequencing Conclusion
Achievements


Photo 1. Fluorescent devices and LB control. A photo of the three devices and an LB control was taken with a help of transillumiantor

  • Successfully built the three devices
  • Sequenced and gel verified the three devices
  • Characterised all the three devices with the OD600 and fluorescence measurements in a Tecan micro-plate reader
  • Reported the results on the Interlab Study page of the iGEM wiki
  • Completed the description of the BBa_I20260 (Device 1)
  • Submitted and send parts: BBa_K1403000 (Device 2) and BBa_K1403001 (Device 3) to the Biobricks registry


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. "

Motivation

iGEM Paris Bettencourt team supported this initiative with pleasure. We strongly believe that this study will help to define standards for BioBrick constructs and measure the difference between different labs and measurement methods. It was also a great training for transformation and ligation techniques for the inexperienced synthetic biologist on the team.




Devices

We built 3 devices (Fig. 1, Fig. 2, Fig. 3) according to Interlab study instructions. First designed in software (Geneious v. 7.0.6). Then made in our wet lab using the Biobrick Distribution Kits and confirmed by electrophoresis gel analytic digestion as well as sequencing. Finally, stocked as a glycerol stock and used for the measurements of GFP expression.

Figure 1. Device 1. Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​


Device 1

Figure 1. Device 1. Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​

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

We followed iGEM Distribution Kit instructions to extract DNA from the Biobrick Plate 2, Well 17F (2012) and then Heat Shock transformed the E. coli colonies that grew in the selective Kanymycin were grown in liquid media and made into glycerol stocks labelled G.22.

Figure 2. Device 2.Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​



Device 2

Our part in the registry: BBa_K1403000

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

We followed the iGEM Distribution Kit instructions to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then Heat Shock transformed E. coli
The colonies that survived after selection in choloamphenicol were cultured overnight. We used 750uL of the liquid cultures for a glycerol stock . The remaining 4.25 mL were used to make minipreps . We measured DNA content with the nanodrop.

Digestion analysis:

  • 5 ug plasmid
  • 5 ul 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 extracted BBa_E0240 with a gel extraction kit. For the plasmid with the promoter we used a PCR purification kit. We 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
We transformed the ligation product following Heat Shock transformation of E. coli. We made liquid cultures of single colonies with the appropriate antibiotic and the next day we prepared a glycerol stock.

Figure 3. Device 3.Geneious version 7.0.6 created by Biomatters. Available from ​http://www.geneious.com/​​



Device 3

Our part in the registry: BBa_K1403001

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

BBa_J23115 + BBa_E0240 (B0032-E0040-B0010-B0012), in the pSB1C3 vector.
Selection marker : Chloramphenicol
Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc (mismatched 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 proceeded 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.


1. Device 1




...GTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc

Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC

Complete sequenced device:

ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAA...



2. Device 2




...AGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc

Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC

Complete sequenced device:

TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGA...



3. Device 3




...GTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA

Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc

Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC

Complete sequence:

GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAA...


Figure 4. 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).

Figure 5. 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).

Figure 6. 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).



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).




Results

Here we present the result of the measurments. The measument has been realised in a comparable growth conditions for all Devices (Fig. 4). We can clearly see that the highest GFP expression levels occures under BBa_J23101 Anderson's strong promoter in a high copy plasmid sPB1C3 (Device 2: BBa_K1403000). The lowest GFP levels occures under very weak Anderson's promoter - mutated J23115 (Device 3: BBa_K1403001) as can be seen in the Fig. 5 & Fig. 6

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