Team:Evry/Interlab Study/Results

From 2014.igem.org

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There were two sub-parts in this study:
There were two sub-parts in this study:
<ul>
<ul>
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<li>The mandatory one: obtain fluorescence data for three specific genetic devices expressing GFP and compare them. (donner les constructions)
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<li>The mandatory one: obtain fluorescence data for three specific genetic devices expressing GFP and compare them.
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  <a id="igem-link" target="_blank" href="http://parts.igem.org/Promoters/Catalog/Anderson " title="Go to iGEM site">(J23100 to J23119)</a>.
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<li>The Extra Credit assignments: we choose to study the entire Anderson library of constitutive prokaryotic promoters <a id="igem-link" target="_blank" href="http://parts.igem.org/Promoters/Catalog/Anderson " title="Go to iGEM site">(J23100 to J23119)</a>.
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<li>The Extra Credit assignments: we choose to study the entire Anderson library of constitutive prokaryotic promoters.
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<br/>
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This library corresponds to 19 constitutive promoters containing some nucleotide mutations on the -35 and the -10, as shown figure x. The idea is to compare the expression strength of promoters according to mutations, from the maximum amount of fluorescence data in order to have significant results.
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This library corresponds to 19 constitutive promoters containing some nucleotide mutations on the -35 and the -10, as shown <a href="https://2014.igem.org/Team:Evry/Interlab_Study/Used_Devices"> Used Biobricks and plasmids </a>.
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The idea is to compare the expression strength of promoters according to mutations, from the maximum amount of fluorescence data in order to have significant results.
</ul>  
</ul>  
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    To test the expression of promoters, they are cloned into the PSB1C3 vector followed by the
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    <a id="igem-link" target="_blank" href="http://parts.igem.org/Part:BBa_E0240" title="Go to iGEM site">BBa_E0240</a>, a GFP generator. Constructions are inserted in E. coli DH5 alpha that grown on LB medium added with the selection agent, chloramphenicol. Our team decided to test the fluorescence with a 
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    <a id="igem-link" target="_blank" href="http://www.tecan.com/platform/apps/product/index.asp?MenuID=1813&ID=1918&Menu=1&Item=21.2.10.1.1 " title="Go to iGEM site">TECAN infiniteM200</a> in 96 well plates.
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    Fluorescence data were analyzed via following formula that come from <a id="igem-link" target="_blank" href="http://www.biomedcentral.com/1752-0509/4/55 " title="Go to iGEM site">2010 De Jong et al </a><i> Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria </i>.
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    <a href="https://static.igem.org/mediawiki/parts/0/05/BerkiGEM2006-Promoters.jpg" class="image"></a>
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    <center><img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/8/88/BMC_formlula.emf.jpg" width="300px;float:left;" class="thumbimage"/><br/><center/><br/>
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  Equation A: A(t) is the corrected absorbance of DH5 alpha with the functional reporter system, Au(t)the uncorrected  absorbance of DH5 alpha with the functional reporter system and Ab(t)the background absorbance corresponding to LB  with chloramphenicol medium absorbance.<br>
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  Equation B: B(t) is the absorbance of the strain carrying the promoterless vector, Iu(t) the uncorrected fluorescence intensity of DH5 alpha with the functional reporter system and Ib(t) the background fluorescence intensity of LB chloramphenicol medium with the DH5 alpha carrying the promoterless vector.<br>
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  </div>
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<h2>Required Devices</h2>
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<h4><font color ="blue">Required Devices</font></h4>
</br>
</br>
<table>
<table>
<tr>
<tr>
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   <td> The three required constructions shown various fluorescence intensity at OD 600 nm = 0.5, as shown on bar chart on the right. The same trend was observed between OD 600 nm = 0.1 to 0.8(Data shown on
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   <td>  
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<a href="https://2014.igem.org/Team:Evry/Interlab_Study/Notebook" >Interlab study notebook</a>). Fluorescence intensity curve profile according to OD 600 nm corresponded to the expected profile for a constitutive promoter (Data shown on
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    <center><img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/7/78/Gaph_final.png" width="500p" class="thumbimage"/><br/></center><br/>
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<a href="https://2014.igem.org/Team:Evry/Interlab_Study/Notebook" >Interlab study notebook</a>).
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</td>
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   <td>
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<br/><div align="right"><img src="https://static.igem.org/mediawiki/2014/7/7d/Interlab_final.png" width="500px"; alt="image not found" /></div>
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<center><img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/7/7d/Interlab_final.png" width="500p" class="thumbimage"/><br/></center><br/>
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</br>
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<br>
</td>
</td>
</tr>
</tr>
</table>
</table>
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<h4>Entire Anderson library of constitutive promoters (J23100-J23119)</h4>
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<div align="justify">
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The three required constructions shown various fluorescence intensity at OD 600 nm = 0.5, as shown on graph and bar chart below.  As shown on corrected GFP fluorescence intensity according to OD 600 nm, the relationship between corrected GFP fluorescence and OD 600 nm is linear that corresponded to the expected profile for a constitutive promoter.
 +
<br>
 +
<br>
 +
Theoritically constructions I20260 and J3101 were similar, constructions and alignments in sillico matched perfectly. The difference between these constructions lies on the assembly way. However we obtained, as some other teams (University of Oxford, Paris-Bettencourt and ITESM-CEM), significant GFP expression difference. There is probably a Biobrick standard  construction problem, which relates to the traited question on our <a href="https://2014.igem.org/Team:Evry/Policy_and_Practices/Philosophy"> Phylosophy </a> subsection.
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</div>
<br/>
<br/>
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<h4><font color ="blue">Entire Anderson library of constitutive promoters (J23100-J23119)</font></h4>
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<br>
<table>
<table>
<tr>
<tr>
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  <td><div class="thumb tnone">
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<td>  
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  <a href="https://static.igem.org/mediawiki/parts/0/05/BerkiGEM2006-Promoters.jpg" class="image">
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    <center><img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/8/8a/Graph_Anderson_all.png" width="500p" class="thumbimage"/><br/></center><br/>
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    <img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/0/05/BerkiGEM2006-Promoters.jpg" width="310px" class="thumbimage"/>
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</td>
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  </a>
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  <td>
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  </div>
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<center><img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/b/b6/Graph_Anderson.png" width="500p" class="thumbimage"/><br/></center><br/>
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  <div align="left">
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  <left> Anderson collection promoter sequences and mutations. </center>
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</td>
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  <td><div class="thumb tnone">
 
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  <a href="URL IMAGE" class="image">
 
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    <img alt="IMAGE" src="URL IMAGE" width="310px" class="thumbimage"/>
 
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  </a>
 
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  </div>
 
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  <div align="left">
 
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  <left> Anderson collection promoter sequences and mutations. </center>
 
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  </div></td>
 
</tr>
</tr>
</table>
</table>
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<div align="justify">
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Our team achieved the 19 constructions of the Anderson library. Constructions were first designed in a software(Geneious v. 6.1.6)and then relized on web lab using iGEM 2014 Distribution kit. Verifications were made by eletrophoresis gel analytic PCR amplification and sequencing. Glycerol stock of 3 colonies per constructions were conserved and used for the measurments of GFP expression.
 +
<br>
 +
Unfortunatly, we did not collect satisfying data for the 19 constructions,although assays were repeated several times. The data profile was repeatable under people, apparatus and strain. Strain carrying promotorless vector seemed to had higher fluorescence intensity than strain containing constructions from the Anderson library, that explains negative curve on Corrected GFP fluorescence intensity according to OD 600 nm graph.
 +
However exploitable data were collected for 5 constructions (100, 104, 105, 107 and 118) for which  the relationship between corrected GFP fluorescence and OD 600 nm was linear that corresponded to the expected profile for a constitutive promoter (See graph Corrected GFP fluorescence intensity according to OD 600 nm). Corrected GFP fluorescence intensity at OD 600 nm = 0.45 of these 5 constructions were presented on bar chart above and compared with <a id="igem-link" target="_blank" href="http://parts.igem.org/Promoters/Catalog/Anderson " title="Go to iGEM site">registry data</a> on table below.
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</div>
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<br>
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<br>
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<center><img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/c/c2/Results_table.png" width="400p" class="thumbimage"/></br></center></br>
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<div align="justify">
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<i>Ratio were calculated dividing constructions corrected GFP fluorescence at 0.45 OD 600 nm by construction 100 corrected GFP fluorescence at 0.45 OD 600 nm.</i>
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</div>
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 +
<br>
 +
<div align="justify">
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Results were similar to registry results for the 100, 105 and 107 constructions contrary to constructions 104 and 118 for which data were significantly divergent.
 +
 +
<br>
 +
<br>
 +
 +
Possible approach to overcome data acquisition problems:
 +
<ul>
 +
<li> Using M9 medium instead of LB medium to minimize the basal medium fluorescence </li>
 +
<li> Using another E. coli strain that has a smaller natural fluorescence</li>
 +
<li> Changing of dilution and gain to find the optimal one</li>
 +
<li> Choosing a TECAN with a lower detection limits to collect data for weak promoters</li>
 +
</ul>
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 +
<br>
 +
<br>
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</div>
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Latest revision as of 01:50, 18 October 2014

IGEM Evry 2014

Interlab study - Aim & Results

The goal of the interlab study is to obtain GFP fluorescence data from the iGEM teams all around the world for about twenty constructions. This will permit to observe the result repeatability across technics, strains and teams.

There were two sub-parts in this study:
  • The mandatory one: obtain fluorescence data for three specific genetic devices expressing GFP and compare them.
  • The Extra Credit assignments: we choose to study the entire Anderson library of constitutive prokaryotic promoters (J23100 to J23119).
    This library corresponds to 19 constitutive promoters containing some nucleotide mutations on the -35 and the -10, as shown Used Biobricks and plasmids . The idea is to compare the expression strength of promoters according to mutations, from the maximum amount of fluorescence data in order to have significant results.

Required Devices


IMAGE

IMAGE


The three required constructions shown various fluorescence intensity at OD 600 nm = 0.5, as shown on graph and bar chart below. As shown on corrected GFP fluorescence intensity according to OD 600 nm, the relationship between corrected GFP fluorescence and OD 600 nm is linear that corresponded to the expected profile for a constitutive promoter.

Theoritically constructions I20260 and J3101 were similar, constructions and alignments in sillico matched perfectly. The difference between these constructions lies on the assembly way. However we obtained, as some other teams (University of Oxford, Paris-Bettencourt and ITESM-CEM), significant GFP expression difference. There is probably a Biobrick standard construction problem, which relates to the traited question on our Phylosophy subsection.

Entire Anderson library of constitutive promoters (J23100-J23119)


IMAGE

IMAGE


Our team achieved the 19 constructions of the Anderson library. Constructions were first designed in a software(Geneious v. 6.1.6)and then relized on web lab using iGEM 2014 Distribution kit. Verifications were made by eletrophoresis gel analytic PCR amplification and sequencing. Glycerol stock of 3 colonies per constructions were conserved and used for the measurments of GFP expression.
Unfortunatly, we did not collect satisfying data for the 19 constructions,although assays were repeated several times. The data profile was repeatable under people, apparatus and strain. Strain carrying promotorless vector seemed to had higher fluorescence intensity than strain containing constructions from the Anderson library, that explains negative curve on Corrected GFP fluorescence intensity according to OD 600 nm graph. However exploitable data were collected for 5 constructions (100, 104, 105, 107 and 118) for which the relationship between corrected GFP fluorescence and OD 600 nm was linear that corresponded to the expected profile for a constitutive promoter (See graph Corrected GFP fluorescence intensity according to OD 600 nm). Corrected GFP fluorescence intensity at OD 600 nm = 0.45 of these 5 constructions were presented on bar chart above and compared with registry data on table below.


IMAGE

Ratio were calculated dividing constructions corrected GFP fluorescence at 0.45 OD 600 nm by construction 100 corrected GFP fluorescence at 0.45 OD 600 nm.

Results were similar to registry results for the 100, 105 and 107 constructions contrary to constructions 104 and 118 for which data were significantly divergent.

Possible approach to overcome data acquisition problems:
  • Using M9 medium instead of LB medium to minimize the basal medium fluorescence
  • Using another E. coli strain that has a smaller natural fluorescence
  • Changing of dilution and gain to find the optimal one
  • Choosing a TECAN with a lower detection limits to collect data for weak promoters