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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<h2>Biology</h2>
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<h1 id="week2">October 10<SUP>th</SUP> to 17<SUP>th</SUP></h1>
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<p>
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Ligation of rposLS amplicons into PSB1C3 and transformation. Screen by PCR, restriction of four clones and sequencing. Construction with fhaLS from pTOPO (EcoRI and PstI) and I13401 (XbaI and EcoRI) and construction with rsmA PCR products. R0011-rsmA-pSB1A2 (construction 31).With R0011 digested by SpeI and PstI and R0011digested by XbaI and PstI.
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</p>
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<p>
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fhaSL-I13401-pSB1AT2 (construction 32) Four positives clones per construction were selected after colony PCR and sent for sequencing. (No restriction screening was performed in order to gain times)
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</p>
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</div>
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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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<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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Sequencing result of construction 29, 30, 31 and 32 are positives. Glycerol stock was performed. Construction from previews construct.
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</p>
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</p>
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                                <p>
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<ul>
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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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<li>R0040-fhaLS-I13401-pSB1A2 (construction 35 : 31 digested by XhoI and PstI and R0040 digested by SalI and PstI)</li>
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                                </p>
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<li>R0040-fhaLS-I13401-pSB6A1 (construction 34: 3A assembly with 31 digested by XbaI and PstI, R0040 digested by EcoRI and SpeI and pSB6A1 digested by EcoRI and PstI)</li>
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                                <p>
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<li>R0011-rsmA-pSB3C5 (construction 33: clone 31 and pSB3C5 digested by EcoRI and PstI)</li>
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CLONING OF THE CONSTRUCTION pCst-RBS-CinR IN pSB3C5:
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                                </p>
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<p>
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The colony PCR screen was performed for construction 33 and 35. One clone for construction 35 and four clones for construction 33 were sent for sequencing. Only one clone for construction 35 appeared fluorescent using the epi-fluorescent microscope visualization. Sequencing was correct for construction 33 and 35
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                                <ul>
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</p>
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<li>Restriction using  EcoRI and Pst1.</li>
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<p>
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Double transformation was performed for rsmA inhibition tests with construction 33 and 35. Flow Cytometry of K253006, constructs 30 and 35 in order to measure the strength of fha and mag. Flow Cytometry of double transformants (33 and 35) in order to measure the effect of rsmA (33) on the translation of GFP mRNA.
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<li>Ligation: standard protocol was performed; Pcst-RBS-CinR was inserted into pSB3C5 plasmids</li>
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</p>
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<p>
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<li>Spreading over Petri dish.</li>
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Test of RBS construct (K256003, 30, 35 and double transformed cells contained construct 30 and 35 with or without IPTG) by FACS and Fusion.
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</p>
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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>
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                                        <li>PCR on clones was performed, 8 clones had the two plasmids.</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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PREPARATION OF THE 96 WELLS PLATE.
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                                        </p>
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                              <ul>
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                                        <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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<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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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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