Team:Paris Bettencourt/Experiments/SinOp

From 2011.igem.org

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<h1>SinOp system experiments</h1>
 
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<h2>Desing and cloning for the KinA</h2>
 
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{| border="1" class="wikitable" style="text-align: center;"
 
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|Sporulation assay with KinA overexpression
 
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|[[File:spor1.jpg|450px|thumb|center|Induction of sporulation after 3h on CDH Medium +4mM IPTG]]
 
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|[[File:spor2.jpg|450px|thumb|center|Induction of sporulation after 3h on CDH Medium +4mM IPTG]]
 
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[[File:control spor.jpg|450px|thumb|center|Negative control of sporulation after 3h on CDH Medium +0mM IPTG]]
 
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<h1>SinOp system experiments</h1>
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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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<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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    <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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<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>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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<h2>Characterization of the sporulation induced by the expression of KinA</h2>
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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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<p>We used a strain holding a KinA gene under the control of a hyperspank promoter. Growing the cell into a synthetic minimal media for for hours, we saw the cell starting soprulating under the microscope after 1h30. Here are the images from the characterization.</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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<td></td>
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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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<br />
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<br />
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<p>In these images we see...
 
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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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== Characterizing the SinI system ==
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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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As explained in the design page
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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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In order to verify the function of SinI, we tested its activity under the Hyperspank promoter. Upon induction, a clear phenotypic change occured where cells turned into chain-like growth regime (fluorescent cellsin image), unlike wild-type cells that kept their regular elongated separated cell phenotype (non-fluorecent cells in image).
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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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<p>You can find the video of our results for the tRNA with IPTG <a href="http://www.youtube.com/watch?v=LIIm0WDBRjM"> here </a> and without IPTG <a href="http://www.youtube.com/watch?v=Yfdv_Ut-kRw">here</a>.</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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=== Preparation of slides ===
 
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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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Two well slides : 1-control (YC164 with IPTG)  2-Mix (both strains with IPTG)
 
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<br>
 
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=== Observation  ===
 
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-37°C Microscopy-<br>
 
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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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* YC164 grows fast (several divisions over the experiments)
 
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* YC227 does not grow at all and keeps its fluorescence pretty well
 
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* YC164 does not change its state (no obvious biofilm formation)
 
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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_left"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs"><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/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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