Team:Warsaw/Stage2

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<h1>Safety</h1>
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<h2>Kill-switch</h2>
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<h3> Kill-switch system: a safety system that maintains stable amounts of <i>E.coli</i> designed for eucaryotic cells transfection and gene delivery </h3>
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<div class="note"> Aim and the basis </div>
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<p>The aim of our kill-switch safety project is to design and measure a system that enables maintaining stable amounts of <i>E.coli</i> during eucaryotic cells transfection.</p>
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<p> Its functionality is based on <a href="http://partsregistry.org/Part:BBa_K299806">MinC protein</a> – a protein that inhibits bacterial cell division. <a href="http://partsregistry.org/Part:BBa_K299806">MinC</a> prevents FtsZ ring formation in the polar region of the cell (see <a href="https://2010.igem.org/Team:Warsaw/Stage2/Background">background</a>). <a href="http://partsregistry.org/Part:BBa_K299806">MinC</a> protein slowers division of <i>E.coli</i> cells but it doesn’t destroy them. In this case, it enables safe delivery of our desired proteins to the inside of eucaryotic cells – in case of leaky expression it doesn’t cause premature bacterial lysis and release of our proteins. The system is universal – it can be used in all bacterial species forming FtsZ ring during cell division (see <a href="https://2010.igem.org/Team:Warsaw/Stage2/Background">background</a>).</p>
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<div class="note"> Measurement </div>
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<p>We performed both dynamic and static measurements (see <a href="https://2010.igem.org/Team:Warsaw/Stage2/Design">design</a> and <a href="https://2010.igem.org/Team:Warsaw/Stage2/Results">results</a>). Dynamic measurement describes optical density (OD) of <i>E.coli</i> and the number of colony forming units (cfu/ml) on agar plates due to IPTG induction time. Static measurement describes OD and cfu/ml under different IPTG concentrations. We used two different negative controls: one carrying <a href="http://partsregistry.org/Part:BBa_K299806">MinC</a> part only on pSB plasmid, IPTG induced, the second carrying our construct without IPTG induction (our control for leaky expression).
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During the experiment we used two different RBSes (<a href="http://partsregistry.org/Part:BBa_B0032">B0032</a> and <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>) for additional regulation of gene expression to study how much different RBSes would influence the general expression of this system (see <a href="https://2010.igem.org/Team:Warsaw/Stage2/Design">design</a>). </p>
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<p> Finally, we observed filamental <i>E.coli</i> cells formation under the <a href="https://static.igem.org/mediawiki/2010/0/08/Filament.jpg">microscope</a>. The observation is consistent with theoretical basis of <a href="http://partsregistry.org/Part:BBa_K299806">MinC</a> function. <a href="http://partsregistry.org/Part:BBa_K299806">MinC</a> stops cell division even tough bacterium can grow and enlarge its size.
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<img src="https://static.igem.org/mediawiki/2010/2/2c/Switch_warsaw.png" align="right" float="right"/></p>
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Latest revision as of 23:25, 26 October 2010

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Kill-switch

Kill-switch system: a safety system that maintains stable amounts of E.coli designed for eucaryotic cells transfection and gene delivery

Aim and the basis

The aim of our kill-switch safety project is to design and measure a system that enables maintaining stable amounts of E.coli during eucaryotic cells transfection.

Its functionality is based on MinC protein – a protein that inhibits bacterial cell division. MinC prevents FtsZ ring formation in the polar region of the cell (see background). MinC protein slowers division of E.coli cells but it doesn’t destroy them. In this case, it enables safe delivery of our desired proteins to the inside of eucaryotic cells – in case of leaky expression it doesn’t cause premature bacterial lysis and release of our proteins. The system is universal – it can be used in all bacterial species forming FtsZ ring during cell division (see background).

Measurement

We performed both dynamic and static measurements (see design and results). Dynamic measurement describes optical density (OD) of E.coli and the number of colony forming units (cfu/ml) on agar plates due to IPTG induction time. Static measurement describes OD and cfu/ml under different IPTG concentrations. We used two different negative controls: one carrying MinC part only on pSB plasmid, IPTG induced, the second carrying our construct without IPTG induction (our control for leaky expression). During the experiment we used two different RBSes (B0032 and B0034) for additional regulation of gene expression to study how much different RBSes would influence the general expression of this system (see design).

Finally, we observed filamental E.coli cells formation under the microscope. The observation is consistent with theoretical basis of MinC function. MinC stops cell division even tough bacterium can grow and enlarge its size.

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