Team:Paris Bettencourt/Experiments/SinOp

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==Results==
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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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=== Preparation of slides ===
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<p>Here is the cloning we made for this system:</p>
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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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Dilution of overnight cultures : YC164 and YC227 (SinOp Design) . <br>
 
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YC227 produces SinI which enhances biofilm formation. If SinI diffuses through the nanotubes we expect to see fluorescence from Peps-gfp construct in the receiver cells. <br>
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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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Two well slides : 1-control (YC164 with IPTG)  2-Mix (both strains with IPTG)
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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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<br>
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=== Observation  ===
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<h2>Characterization of the receiver strain from Veening</h2>
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-37°C Microscopy-<br>
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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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We observed the plate with TRANS and YFP-filter settings on the Old Zeiss. Unfortunately, this microscope gets quickly out of focus so we need to take each picture manually. As expected, YC227 was producing GFP at the begining of the experiment, while YC164 was not. Our observations over 4 hours are the following:
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<h2>Characterizing the KinA emitter</h2>
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* YC164 grows fast (several divisions over the experiments)
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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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* YC227 does not grow at all and keeps its fluorescence pretty well
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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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* YC164 does not change its state (no obvious biofilm formation)
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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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* After letting the plate overnight we still see some florescence but not as if YC164 changed its fluorescence status.
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[[File:0905_SinOp_trans1.jpg|500px|thumb|center|TRANS at t=0min]]
 
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[[File:0905_SinOp_GFP1.jpg|500px|thumb|center|GFP at t=0min]]
 
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[[File:0905_SinOp_trans14.jpg|500px|thumb|center|TRANS at t=90min]]
 
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[[File:0905_SinOp_GFP14.jpg|500px|thumb|center|GFP at t=90min]]
 
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=== Our conclusions ===
 
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We might have poorly chosen the exact phase growth or our timing when plating the strains. Nanotube formation seems to require a more tightly packed colony. We will try this tomorrow. Biofilm formation is usually observed at lower temperatures and it could mean we will never see anything with this design (be reminded that we did not do any cloning for this construction, we only add to reuse strains from another scientific team).
 
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<div id="scroll_right"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs"><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/Designs">To the designs!</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.


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