Team:Trieste/parts/8

From 2012.igem.org

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<h2>Assembly</h2>
<h2>Assembly</h2>
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The BioBrick BBa_K314200 coding for the Tse2 toxin was assembled with the weak BBa_B0031 RBS and with the BBa_B0015 terminator. This composite was then cloned under the inducible promoter T5 LacO ( that we submitted as BioBrick BBa_K875002 ) in order to be able to follow the toxin activity when expressed in <i>E.coli</i>.
<center><img src="https://static.igem.org/mediawiki/2012/7/7f/Tse2ts.gif" width="500px"/></center>
<center><img src="https://static.igem.org/mediawiki/2012/7/7f/Tse2ts.gif" width="500px"/></center>
<h2>Results</h2>
<h2>Results</h2>
<center><img src="https://static.igem.org/mediawiki/2012/c/c0/TsTse2021.jpg" width="600px"/></center></br>
<center><img src="https://static.igem.org/mediawiki/2012/c/c0/TsTse2021.jpg" width="600px"/></center></br>
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<p>M15 bacteria cells (containing the pREP4 plasmid coding for the Lac repressor)  are transformed with this Biobrick that resides on pSB1C3 plasmid.</br>
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<p>M15 bacterial cells (containing the pREP4 plasmid coding for the Lac repressor)  are transformed with this Biobrick that resides on pSB1C3 plasmid.</br>
</br>
</br>
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After an over night liquid culture (LB broth) the OD600 absorbance is recorded and the culture is diluted to 0,1 and the batch is splitted into 6 flask – 20ml each. Three of these are induced with IPTG 1mM and the others are not induced.</br>
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After an over night liquid culture (LB broth) the O.D.<sub>600</sub> absorbance was recorded and the culture was diluted to O.D.<sub> 600</sub>=0,1 and splitted into 6 flask – 20ml each. Three of these were induced with IPTG 1mM and the others were not induced.</br>
</br>
</br>
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The OD 600 is then recorded each hour.</br>
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The O.D.<sub>600</sub> was then recorded every hour.</br>
</br>
</br>
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It is possible to observe that not induced cells grow up to OD 1, instead the induced ones do not growth during the time, demonstrating the inducible promoter and toxin efficiency.</br>
+
It is possible to observe that the not induced cells grew up to O.D.<sub>600</sub> = 1, while the induced ones did not grow, demonstrating the efficiency of both the inducible promoter and the toxin.</br>
</p>
</p>
<br/>  
<br/>  
<br/>  
<br/>  
<h2>Looking forward</h2>
<h2>Looking forward</h2>
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<p> In the final system, the toxin should be under the control of the T5 Cumate Operator (BBa_K875001).
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<p> In the final system, the toxin should be under the control of the T5 Cumate Operator (BBa_K875001). You can find the results on <a href="https://2012.igem.org/Team:Trieste/project/mainres"> Main Results page</a>, in the suicide system section.
</p>
</p>
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                 <li><a href="https://2012.igem.org/Team:Trieste/parts/6">BBa_K875006 - PelB scFv</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/6">BBa_K875006 - PelB scFv</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/7">BBa_K875007 - PelB SIP </a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/7">BBa_K875007 - PelB SIP </a></li>
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                 <li><a href="https://2012.igem.org/Team:Trieste/parts/8">BBa_K875008 - Tse2 Toxin</a></li>
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                 <li class="select"><a href="https://2012.igem.org/Team:Trieste/parts/8">BBa_K875008 - Tse2 Toxin</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/9">BBa_K875009 - LL 37</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/9">BBa_K875009 - LL 37</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/10">BBa_K875020 - Glucosidase</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/10">BBa_K875020 - Glucosidase</a></li>

Latest revision as of 18:02, 26 October 2012

BBa_K875008

More

Description

The Tse2 protein is a toxin component of a toxin-immunity system and arrests the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 has no effect on eukaryotic cell.


Assembly

The BioBrick BBa_K314200 coding for the Tse2 toxin was assembled with the weak BBa_B0031 RBS and with the BBa_B0015 terminator. This composite was then cloned under the inducible promoter T5 LacO ( that we submitted as BioBrick BBa_K875002 ) in order to be able to follow the toxin activity when expressed in E.coli.

Results



M15 bacterial cells (containing the pREP4 plasmid coding for the Lac repressor) are transformed with this Biobrick that resides on pSB1C3 plasmid.

After an over night liquid culture (LB broth) the O.D.600 absorbance was recorded and the culture was diluted to O.D. 600=0,1 and splitted into 6 flask – 20ml each. Three of these were induced with IPTG 1mM and the others were not induced.

The O.D.600 was then recorded every hour.

It is possible to observe that the not induced cells grew up to O.D.600 = 1, while the induced ones did not grow, demonstrating the efficiency of both the inducible promoter and the toxin.



Looking forward

In the final system, the toxin should be under the control of the T5 Cumate Operator (BBa_K875001). You can find the results on Main Results page, in the suicide system section.


Link to the Registry


Università degli studi di Trieste ICGEB Illy Fondazione Cassa di Risparmio
iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012
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