Team:Grenoble/Notebook/October

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<h1 id="week1">September 2<SUP>nd</SUP> to 9<SUP>th</SUP></h1>
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<h1 id="week1">October 2<SUP>nd</SUP> to 9<SUP>th</SUP></h1>
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                            <p>
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Amsterdam regional jamboree.
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                            </p>
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<div class="blocbackground">
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<h1 id="week2">October 10<SUP>th</SUP> to 17<SUP>th</SUP></h1>
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<h1 id="week2">September 10<SUP>th</SUP> to 17<SUP>th</SUP></h1>
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<div class="blocbackground">
<div class="blocbackground">
<h2>Biology</h2>
<h2>Biology</h2>
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                        <div class="noindent">
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<p>
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The aim of our experiments is to demonstrate the coloration, for that we have done different experiments:
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</p>
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                                <p>
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The goal is to introduce the constructions (pcst-RBS-CinR) and (pCin-lycopene) on the same bacterium, the constructions are supported on pSB3C5 and pSB1A2 respectively., by adding externel AHL molecule, this latter can enter in the bacterium and forms with CinR molecules complex that allows the activation of Lycopene production, after few times we can see the red color.
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                                </p>
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                                <p>
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CLONING OF THE CONSTRUCTION pCst-RBS-CinR IN pSB3C5:
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                                </p>
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                                <ul>
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<li>Restriction using  EcoRI and Pst1.</li>
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<li>Ligation: standard protocol was performed; Pcst-RBS-CinR was inserted into pSB3C5 plasmids</li>
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<li>Spreading over Petri dish.</li>
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<li>Better cloning rate (New stock of biobrick Miniprep)</li>
 +
                               
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<li>Do checking, PCR cheking.</li>
 +
                               
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<li>To confirm the result gel checking was performed.</li></ul>
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                                        <p>
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pCin-Lycopene CONSTRUCTION IN pSB1A2 PREPARATION
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                                        </p>
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                              <ul>
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                                        <li> Culture overnight (the biobrick Miniprep was prepared).</li></ul>
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                                        <p>
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Double transformation (introduction of the two plasmids contain pCst-RBS-CinR and  pCin-Lycopene respectively).
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                                        </p>
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                              <ul>
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                                        <li>Spreading over Petri dish containing double resistances Ch and Amp.
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                                        <li>Good results, we choose 15 clones.</li>
 +
                                        <li>PCR on clones was performed, 8 clones had the two plasmids.</li>
 +
                                        <li>To confirm the result gel checking was performed.</li></ul>
 +
                                        <p>
 +
PREPARATION OF THE 96 WELLS PLATE.
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                                        </p>
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                              <ul>
 +
                                        <li>We have tested our 8 clones with three different concentration of AHL (1mM, 0,5mM and 0,2mM).</li></ul>
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 +
</div>
</div>
</div>
</div>
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<h1 id="week3">October 18<SUP>th</SUP> to 25<SUP>th</SUP></h1>
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</div>
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<div class="blocbackground">
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<h2>Biology</h2>
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<div class="noindent">
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  <h3>Characterization of the toggle switch</h3>
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  <p>Cells are grown at 37°C for 24h with either 500 ng/ml aTC, 2 mM IPTG or without inducers. At 7h and 15h, cells are washed and diluted in a fresh medium. Then cells are grown for an hour without inducers. By microscopy, we could see cells grown with aTc don't express gfp. Cells grown with IPTG or without inducers express gfp.</p>
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  <center>
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  <a href="https://static.igem.org/mediawiki/2011/a/a1/Figure_preculture.png"><img src="https://static.igem.org/mediawiki/2011/a/a1/Figure_preculture.png" ></a>
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  </center>
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  <center>
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  <a href="https://static.igem.org/mediawiki/2011/9/98/Figure_oct3.png"><img src="https://static.igem.org/mediawiki/2011/9/98/Figure_oct3.png" class="centerwide"/></a>
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  <div class="legend">Demonstration of the switching dynamics and long term stability experiment</div>
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  </center>
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  <center>
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  <a href="https://static.igem.org/mediawiki/2011/c/cb/Figure_oct2.png"><img src="https://static.igem.org/mediawiki/2011/c/cb/Figure_oct2.png" class="centerwide"/></a>
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  <div class="legend">Toggle switching time experiment</div>
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  </center>
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</div>
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Latest revision as of 03:58, 29 October 2011

Grenoble 2011, Mercuro-Coli iGEM


October 2nd to 9th

Amsterdam regional jamboree.

October 10th to 17th

Biology

The aim of our experiments is to demonstrate the coloration, for that we have done different experiments:

The goal is to introduce the constructions (pcst-RBS-CinR) and (pCin-lycopene) on the same bacterium, the constructions are supported on pSB3C5 and pSB1A2 respectively., by adding externel AHL molecule, this latter can enter in the bacterium and forms with CinR molecules complex that allows the activation of Lycopene production, after few times we can see the red color.

CLONING OF THE CONSTRUCTION pCst-RBS-CinR IN pSB3C5:

  • Restriction using EcoRI and Pst1.
  • Ligation: standard protocol was performed; Pcst-RBS-CinR was inserted into pSB3C5 plasmids
  • Spreading over Petri dish.
  • Better cloning rate (New stock of biobrick Miniprep)
  • Do checking, PCR cheking.
  • To confirm the result gel checking was performed.

pCin-Lycopene CONSTRUCTION IN pSB1A2 PREPARATION

  • Culture overnight (the biobrick Miniprep was prepared).

Double transformation (introduction of the two plasmids contain pCst-RBS-CinR and pCin-Lycopene respectively).

  • Spreading over Petri dish containing double resistances Ch and Amp.
  • Good results, we choose 15 clones.
  • PCR on clones was performed, 8 clones had the two plasmids.
  • To confirm the result gel checking was performed.

PREPARATION OF THE 96 WELLS PLATE.

  • We have tested our 8 clones with three different concentration of AHL (1mM, 0,5mM and 0,2mM).

October 18th to 25th

Biology

Characterization of the toggle switch

Cells are grown at 37°C for 24h with either 500 ng/ml aTC, 2 mM IPTG or without inducers. At 7h and 15h, cells are washed and diluted in a fresh medium. Then cells are grown for an hour without inducers. By microscopy, we could see cells grown with aTc don't express gfp. Cells grown with IPTG or without inducers express gfp.

Demonstration of the switching dynamics and long term stability experiment
Toggle switching time experiment
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