Team/CINVESTAV-IPN-UNAM MX/Construction.htm

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    <li class="active"><a href="promoterselection.htm">Promoter selection </a></li>
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    <h1><em>Construction!</em></h1>
 +
    <p id="text2"> iGEM PROMOTER CHARACTERIZATION</p>
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 +
    <p>We need to use a medium strength promoter for our constructions, so we characterize different promoters  constitutive belonging to the isolated family from Anderson collection (J23101, J23102, J23104, J23107, J23108, J2311, and J23115) these promoters were assemble with GFP to determine promoter strength, using Victor X3 Multilabel Plate Reader.<br>
 +
      <br>
 +
      <img src="https://static.igem.org/mediawiki/2012/e/e8/Pfigura1.jpg" alt="figura1" width="570" height="180"></p>
 +
    <div align="center"><img src="https://static.igem.org/mediawiki/2012/0/03/Pfigura2.jpg" alt="figura2" width="394" height="179"><br>
 +
      <br>
 +
  <img src="https://static.igem.org/mediawiki/2012/3/39/PFigura3.jpg" alt="Figura3" width="566" height="328">   </div>
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    <br>
 +
    <div align="center"></div>
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    <br>
 +
    <p id="text2"> Result</p>
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    <p>The characterization of the BioBricks shown in the following table:</p>
 +
     <div align="center"><img src="https://static.igem.org/mediawiki/2012/0/02/PFigura4.jpg" alt="Figura4" width="349" height="157"></div>
 +
    <br>
 +
    <img src="https://static.igem.org/mediawiki/2012/5/5b/PFigura5.jpg" alt="Figura5" width="282" height="477" class="left">
 +
    <p>&nbsp;</p>
 +
    <p>&nbsp;</p>
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    <p>&nbsp;</p>
 +
    <p>&nbsp;</p>
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    <p>&nbsp;</p>
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    <p>The constructions that were made, were confirmed by double digestions. The following image shows a gel double digestion with XbaI and SpeI for confirmatory promoter constructs:</p>
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    <br>
 +
     <br>
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     <br>
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    <br>
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<p>The constructions were cultivated in LB petri dishes with chloramphenicol and they were observated at UV light after it’s incubation.</p>
 +
<div align="center">
 +
  <p><img src="https://static.igem.org/mediawiki/2012/7/7a/PFigura6.jpg" alt="Figura6" width="263" height="247" class="left"><img src="https://static.igem.org/mediawiki/2012/e/e4/PFigura7.jpg" alt="Figura7" width="249" height="248" > <img src="https://static.igem.org/mediawiki/2012/0/0b/PFigura8.jpg" width="397" height="384"></p><br>
</div>
</div>
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<div id="bodys_r1_c3">
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<p>In the following graphs there is shown the GFP expression in function of th time and the realtive promoter force. </p><img src="https://static.igem.org/mediawiki/2012/7/72/PFigura9.jpg" alt="Figura9" width="563" height="345">
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<p><br>
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<div class="clearFloat"></div>
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  With the previous results of the characterization of the promoters there is concluded that the promoter J23107, is the strongest because it produces more RPUs.<br><br>
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<div id="bodys_r2_c2"></div>
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After characterization, J23104 promoter was chosen for construction of the system of regulation of light and oxygen dependent.</p>
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<div id="bodys_r2_c3">
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<div align="center"><img src="https://static.igem.org/mediawiki/2012/0/09/PFigura10.jpg" alt="Figura10" width="567" height="419"> </div>
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    <div class="Text">
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  </div>
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  <br>
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      <p><strong>CHARACTERIZATION OF IGEM BIOPARTS DISTRIBUTION</strong></p>
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  <div id="sidebar">
-
      <p>As part of the points required to perform well in  2012 iGEM competition we are asked to characterize some existing biopartes on  the record, so we decided to make the characterization of constitutive promoters belonging to the family isolated from a small combinatorial library (J23101 , J23102, J23104, J23107, J23108, J2311, and J23115) which were attached to GFP  to determine promoter activity, using the equipment Victor X3 Multilabel Plate Reader.</p>
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<p><img src="https://static.igem.org/mediawiki/2012/3/3f/Co1.jpg" width="640" height="186" /></p>
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      <h2>Promoter Selection </h2>
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<p>Fig 1. General scheme of one construction of the promoter expressing GFP</p>
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        <li><a href="Construction.htm" target="_parent">Construction</a></li>
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<p><img src="https://static.igem.org/mediawiki/2012/0/0b/Co2.jpg" width="640" height="470" /></p>
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        <li><a href="Metods.htm">Methods</a></li>
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<p>Fig 2. Final construction</p>
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      </ul>
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<p>&nbsp;</p>
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  <div style="clear: both;">&nbsp;</div>
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Latest revision as of 03:34, 27 October 2012

Rho

Construction!

iGEM PROMOTER CHARACTERIZATION

We need to use a medium strength promoter for our constructions, so we characterize different promoters constitutive belonging to the isolated family from Anderson collection (J23101, J23102, J23104, J23107, J23108, J2311, and J23115) these promoters were assemble with GFP to determine promoter strength, using Victor X3 Multilabel Plate Reader.

figura1

figura2

Figura3


Result

The characterization of the BioBricks shown in the following table:

Figura4

Figura5

 

 

 

 

 

The constructions that were made, were confirmed by double digestions. The following image shows a gel double digestion with XbaI and SpeI for confirmatory promoter constructs:





The constructions were cultivated in LB petri dishes with chloramphenicol and they were observated at UV light after it’s incubation.

Figura6Figura7


In the following graphs there is shown the GFP expression in function of th time and the realtive promoter force.

Figura9


With the previous results of the characterization of the promoters there is concluded that the promoter J23107, is the strongest because it produces more RPUs.

After characterization, J23104 promoter was chosen for construction of the system of regulation of light and oxygen dependent.

Figura10

 

Rhodofactory 2012

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