Team:Paris Bettencourt/Experiments/SinOp

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<h1>SinOp</h1>
 
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<p>You can find the details of the experiments on the <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/09/05/">notebook</a>.</p>
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<h1>SinOp system experiments</h1>
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<h2>Abstract</h2>
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<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>Results for the SinOp system:
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<ul>
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    <li>We successfully BioBricked KinA (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K606046">BBa_K606046</a>) into PSB1C3, improving preexisting biobrick <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K144010">BBa_K144010</a> constructs and sent them to the registry</li>
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<li>We showed that our receptor is a good reporter for sporulation and characterized it.</li>
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<li>We were able to express kinA  and trigger sporulation in rich medium in exponential phase.</li>
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</ul></p></b></div>
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<h2>Design overview</h2>
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<center><img src="https://static.igem.org/mediawiki/2011/1/18/Sinop_principle.jpg"></center>
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<center><p>SinOp system</p></center>
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<p>More information on the design <a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp">here</a>.</p>
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<h2>Parts and biobrick system construction</h2>
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<p>Here is the cloning we made for this system:</p>
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<br />
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<center><a href="https://static.igem.org/mediawiki/2011/8/83/1028_Cloning_plans_kinA.png"><img src="https://static.igem.org/mediawiki/2011/8/83/1028_Cloning_plans_kinA.png"></a></center>
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<p>We suceeded in recovering the KinA gene from the non biobricked plasmid synthetized de novo by the 2009 Newcastle team, and cloned it into a standard biobrick plasmid, pSB1C3. Then we cloned this gene in front of the pVeg-SpovG (K143051) promoter + RBS. These two constructs had bees sended to the registry into pSB1C3.</p>
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<p>This construct has been cloned right away into a replicative plasmid for subtilis and transformed, and we are caracterizing it at the moment.</p>
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<h2>Characterization of the receiver strain from Veening</h2>
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<center> <img src="https://static.igem.org/mediawiki/2011/1/15/Zdjzpojdapjdz.jpg" style="width:600px;"> </center><center>
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<i>SpoIIA-gfp sporulation reporter induced in sporulation medium (CDH) strain from Veening and al 2006. </i></center>
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<p>GFP gene is under SpoIIA promoter. SpoIIA gene is express in sporulation process downstream of the SinOperon. Under starvation conditions, cells sporulate and then express the gfp.</p>
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<center><img src="https://static.igem.org/mediawiki/2011/9/9b/Qdzqd.png" style="width:600px"></center><center>
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<i>Quantification of fluorescence during sporulation. Cells that are sporulating express gfp under the spoIIA promoter</i></center>
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<p>We also measured fluorescence kinetics by spectrophotometry  (Tecan®). </p>
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<h2>Characterizing the KinA emitter</h2>
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<center><img src="https://static.igem.org/mediawiki/2011/7/79/Dqs.jpg" style="width:600px"></center><center>
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<i>Artificial induction of sporulation on LB agar with IPTG in exponential phase at 37 phase.</i></center>
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<p>We overexpressed kinA, in SB168, that triggered sporulation in exponential phase at 37 degrees.  Under microscope, we can observe many of cells harboring small black spots, which are characteristic of sporulation.</p>
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<div id="scroll_right"><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff"><img src="https://static.igem.org/mediawiki/2011/e/e0/Arrow-right-big.png" style="width:100%;"></a><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff">GFP diffusion experiment</a></div>
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<div id="scroll_left"><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp"><img src="https://static.igem.org/mediawiki/2011/0/0a/Arrow-left-big.png" style="width:100%;"></a><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp">SinOp design</a></div>
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Latest revision as of 02:47, 29 October 2011

Team IGEM Paris 2011

SinOp system experiments

Abstract

Results for the SinOp system:

  • We successfully BioBricked KinA (BBa_K606046) into PSB1C3, improving preexisting biobrick BBa_K144010 constructs and sent them to the registry
  • We showed that our receptor is a good reporter for sporulation and characterized it.
  • We were able to express kinA and trigger sporulation in rich medium in exponential phase.

Design overview

SinOp system

More information on the design here.

Parts and biobrick system construction

Here is the cloning we made for this system:





We suceeded in recovering the KinA gene from the non biobricked plasmid synthetized de novo by the 2009 Newcastle team, and cloned it into a standard biobrick plasmid, pSB1C3. Then we cloned this gene in front of the pVeg-SpovG (K143051) promoter + RBS. These two constructs had bees sended to the registry into pSB1C3.

This construct has been cloned right away into a replicative plasmid for subtilis and transformed, and we are caracterizing it at the moment.

Characterization of the receiver strain from Veening

SpoIIA-gfp sporulation reporter induced in sporulation medium (CDH) strain from Veening and al 2006.

GFP gene is under SpoIIA promoter. SpoIIA gene is express in sporulation process downstream of the SinOperon. Under starvation conditions, cells sporulate and then express the gfp.

Quantification of fluorescence during sporulation. Cells that are sporulating express gfp under the spoIIA promoter

We also measured fluorescence kinetics by spectrophotometry (Tecan®).

Characterizing the KinA emitter

Artificial induction of sporulation on LB agar with IPTG in exponential phase at 37 phase.

We overexpressed kinA, in SB168, that triggered sporulation in exponential phase at 37 degrees. Under microscope, we can observe many of cells harboring small black spots, which are characteristic of sporulation.


Retrieved from "http://2011.igem.org/Team:Paris_Bettencourt/Experiments/SinOp"