Team:TU Eindhoven/Safety/Kill Switch

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                  <h2>Our Lab</h2>
 
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                  <p>The laboratory of Chemical Biology is located in the building Helix on the TU Eindhoven campus (see <a href='#Fig1'>Figure 1</a> for a plan). This laboratory is part of the department of Biomedical Engineering. The laboratory of Chemical Biology consists of two laboratories. The first one is a ML-I laboratory with biosafety level 1 (WHO BSL 1). Most work at the ML-I lab involves recombinant expression of proteins in E. coli or yeast expression systems, and the application of bacteriophages for phage display. The second one is a ML-II laboratory (cell lab) with biosafety level 2 (WHO BSL 2). Work in the ML-II lab typically involves the use of immortalized cell lines, but it can also involve tissues or primary cells from animals or humans.</p>
 
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/a/ad/TU_Eindhoven_Laboratory.png" class="image_wrapper image_fr" width="1085">
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<figure style="float:right;margin-right:0;">
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. Plan of the Laboratory of Chemical Biology.</p>
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/7/7c/TU_Eindhoven_Kill_Switch.jpg" width="400" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;">
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<figcaption style="font-size:18px;color:#CCCCCC;">Figure 1: Kill switch overview.</figcaption>
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</figure>
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<h3>Safety Equipment</h3>
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                  <h2>Kill Switch</h2>
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<p>The basic protection equipment that we use in the wet lab are lab coats, safety goggles, and gloves. Lab coats and safety glasses are used always and everywhere in the lab (ML-I and ML-II). Gloves (mostly latex) are used when handling chemicals and for working DNase free. In the cell lab (ML-II) they are also used for working with biological materials in the biosafety cabinets. Gloves (nitrile) are used for handling chemicals that pass through latex or for persons allergic to latex. Cryo-gloves and heat-resistant gloves are used for handling cold and hot materials respectively. Also when working with hazardous or toxic fumes we use the fume hood.</p>
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/e/ed/TU_Eindhoven_Safety_Gloves.JPG" class="image_wrapper image_fr" width="1085">
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                  <p>The kill switch idea is not really necessary for Click Coli as already stated in the <a href="https://2014.igem.org/Team:TU_Eindhoven/Safety/Biosafety">Biosafety page</a>.  
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/d/d5/TU_Eindhoven_Safety_Rest.JPG" class="image_wrapper image_fr" width="1085">
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If gene transfer occurs, it still misses the clickable DBCO functionalized molecules groups and would therefore miss the protective coating. However our idea is a fundamental tool and can thus be used in other future iGEM projects. Therefore it is important for other teams to think about a functional kill switch for their own project when using our tool, because in our case Click Coli does not necessarily need a kill switch system.
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<p style="font-size:18px;color:#CCCCCC;">Figure 2. Figure safety equipment</p>
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<br><br>
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However we still thought about a kill switch system. This system will be active after the bacteria fulfilled their function. This will limit the time the bacteria is active, so there will be less time to adapt or mutate.
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<br><br>
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How does it work? First, the bacterium is induced with IPTG outside the human body. The plasmid in the bacteria has to be IPTG inducible. The IPTG can turn on an IPTG sensitive promotor and then turns up the lambda suppressor (CI) concentration in the cell which will increase exponentially [1].
 +
A Spo0A gene can be implemented behind a promotor that is sensitive for CI. The toggle will activate Spo0A when it reaches the set concentration of CI.
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</p>
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<h3>Working Equipment</h3>
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<h3>Timer</h3>
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<p>We used a lot of equipment during our project, but the most important equipment are standard equipment like pipettes and pipettes tips to measure and transport liquids, but also the Bunsen Burner to work sterile and the centrifuge to spin the samples. Also other equipment is used like the PCR machine for DNA amplification and gel electrophoresis machine for gel extraction.</p>
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<p>An increase of phosphorylation of Spo0A (Spo0A<sup>P</sup>) can cause pulses with an increase in amplitude within every pulse [2].  When reaching a certain concentration of Spo0A<sup>P</sup>, a Spo0A<sup>P</sup> sensitive promotor will initiate the cell death.</p>
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<img id='Fig3' src="https://static.igem.org/mediawiki/2014/1/19/TU_Eindhoven_Equipment_1.JPG" class="image_wrapper image_fr" width="1085">
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<figure style="float:left;margin-left:0;">
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<img id='Fig3' src="https://static.igem.org/mediawiki/2014/2/28/TU_Eindhoven_Equipment_2.JPG" class="image_wrapper image_fr" width="1085">
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/d/d9/TU_Eindhoven_Kill_Switch2.png" width="250" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;">
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<p style="font-size:18px;color:#CCCCCC;">Figure 3. Figure working equipment</p>
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<figcaption style="font-size:18px;color:#CCCCCC;">Figure 2. Phosphorelay gene circuit <br> controlling sporulation initiation.<br> Adapted from Levine et al. (2012).</figcaption>
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</figure>
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<img id='Fig4' src="https://static.igem.org/mediawiki/2014/1/11/TU_Eindhoven_Safety1.jpg" class="image_wrapper image_fr" width="1085">
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<h3>Spo0A<sup>P</sup></h3>
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<p style="font-size:18px;color:#CCCCCC;">Figure 4. Open top bench with amongst other pipets , pipets tips, centrifuge, Bunsen burner and PCR machine.</p>
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<p>Controlling Spo0A activation includes a lot of interactions shown in <a href="#Fig2">Figure 2</a>. Histidine kinases including KinA, transfer phosphates to Spo0A and autophosphates Spo0A through a phosphorelay consisting of Spo0B and Spo0F [2]. However the total level of Spo0A<sup>P</sup> is reduced by phosphates for instance phosphates from the phosphorelay  are drained by rap through Spo0F and Spo0E dephosphorylates Spo0A<sup>P</sup> [2]. A high level of Spo0A<sup>P</sup> can induce sporulation in <i>B. subtilis</i> [2]. But in our case a high level of Spo0A<sup>P</sup> will be necessary to initiate cell death.
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</p>
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<img id='Fig5' src="https://static.igem.org/mediawiki/2014/0/0b/TU_Eindhoven_Safety2.jpg" class="image_wrapper image_fr" width="1085">
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<h3>Pulses</h3>
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<p style="font-size:18px;color:#CCCCCC;">Figure 5. Gel electrophoresis  and SDS-page place.</p>
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<p>The increase in amplitude of pulses is caused by a positive feedback loop [2]. The Spo0A activity pulses activate kinase transcription and this will increase the amplitude of the Spo0A pulses and an increase in kinase activity increases de Spo0A activity pulses shown in <a href="#Fig3">Figure 3</a> [2].</p>
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<img id='Fig6' src="https://static.igem.org/mediawiki/2014/e/e9/TU_Eindhoven_Safety3.jpg" class="image_wrapper image_fr" width="1085">
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<figure style="float:right;margin-right:0;">
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<p style="font-size:18px;color:#CCCCCC;">Figure 6. Storage place for chemicals.</p>
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<img id='Fig3' src="https://static.igem.org/mediawiki/2014/a/ac/TU_Eindhoven_Kill_Switch3.png" width="400" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;">
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<figcaption style="font-size:18px;color:#CCCCCC;">Figure 3. Here is shown that in each cycle the peak <br> amplitude (gray) increases. In green the pulse growth <br>  in a hypothetical trajectory is shown.<br>  Adapted from Levine et al. (2012).</figcaption>
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</figure>
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<img id='Fig7' src="https://static.igem.org/mediawiki/2014/b/b2/TU_Eindhoven_Safety4.png" class="image_wrapper image_fr" width="1085">
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<h3>Conclusion</h3>
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<p style="font-size:18px;color:#CCCCCC;">Figure 7. High speed centrifuge and other lab materials.</p>
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<p>This kill switch idea is not yet complete for our project and is still a concept. One issue is that the Phosphorelay gene circuit controlling sporulation initiation is found in <i>B. subtilis</i>, which is a gram positive bacterium and not in <i>E. coli</i> which is a gram negative bacterium. Therefore it is hard to implement this system in <i>E. coli</i>. Another issue is that our plasmids are IPTG induced just like this kill switch, which means that that the timer would start as soon as we introduce IPTG for protein expression.
 +
<br><br>
 +
When implementing the zwitterionic antifouling protein, gene transfer could occur which could be an issue and therefore we thought about an existing kill switch idea that is more probable.
 +
The idea is the <a target="_blank" href="https://2011.igem.org/Team:Imperial_College_London/Project_Gene_Overview">Gene Guard system (iGEM Imperial College London 2011)</a> which prevents horizontal gene transfer and therefore prevent the exchange of genetic material. A plasmid contains a gene that codes for holin that attaches to the inner membrane and a gene that code for endolysin which causes cell lysis. Antiholin will be integrated in our plasmid and will inhibit holin. This will cause no pores formation in the inner membrane and the endolysin cannot enter the periplasm to break down the outer membrane. This means that the bacteria always need the plasmid with antiholin sequence and the plasmid that encodes for endolysin and holin or else cell lysis occurs in case of plasmid exchange.
 +
</p>
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 +
<h4>Bibliography</h4>
 +
<p>[1] D. Huang, W.J. Holtz, M.M Maharbiz. Journal of Biological Engineering. 6:9 (2012). doi: 10.1186/1754-1611-6-9
 +
<br><br>
 +
[2] J.H. Levine, M.E. Fontes, J. Dworkin, M.B. Elowitz. Pulsed Feedback Defers Cellular Differentiation. PLoS biology. 10:e1001252 (2012). doi:10.1371/journal.pbio.1001252
 +
</p>
</div>
</div>

Latest revision as of 00:45, 18 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Figure 1: Kill switch overview.

Kill Switch

The kill switch idea is not really necessary for Click Coli as already stated in the Biosafety page. If gene transfer occurs, it still misses the clickable DBCO functionalized molecules groups and would therefore miss the protective coating. However our idea is a fundamental tool and can thus be used in other future iGEM projects. Therefore it is important for other teams to think about a functional kill switch for their own project when using our tool, because in our case Click Coli does not necessarily need a kill switch system.

However we still thought about a kill switch system. This system will be active after the bacteria fulfilled their function. This will limit the time the bacteria is active, so there will be less time to adapt or mutate.

How does it work? First, the bacterium is induced with IPTG outside the human body. The plasmid in the bacteria has to be IPTG inducible. The IPTG can turn on an IPTG sensitive promotor and then turns up the lambda suppressor (CI) concentration in the cell which will increase exponentially [1]. A Spo0A gene can be implemented behind a promotor that is sensitive for CI. The toggle will activate Spo0A when it reaches the set concentration of CI.

Timer

An increase of phosphorylation of Spo0A (Spo0AP) can cause pulses with an increase in amplitude within every pulse [2]. When reaching a certain concentration of Spo0AP, a Spo0AP sensitive promotor will initiate the cell death.

Figure 2. Phosphorelay gene circuit
controlling sporulation initiation.
Adapted from Levine et al. (2012).

Spo0AP

Controlling Spo0A activation includes a lot of interactions shown in Figure 2. Histidine kinases including KinA, transfer phosphates to Spo0A and autophosphates Spo0A through a phosphorelay consisting of Spo0B and Spo0F [2]. However the total level of Spo0AP is reduced by phosphates for instance phosphates from the phosphorelay are drained by rap through Spo0F and Spo0E dephosphorylates Spo0AP [2]. A high level of Spo0AP can induce sporulation in B. subtilis [2]. But in our case a high level of Spo0AP will be necessary to initiate cell death.

Pulses

The increase in amplitude of pulses is caused by a positive feedback loop [2]. The Spo0A activity pulses activate kinase transcription and this will increase the amplitude of the Spo0A pulses and an increase in kinase activity increases de Spo0A activity pulses shown in Figure 3 [2].

Figure 3. Here is shown that in each cycle the peak
amplitude (gray) increases. In green the pulse growth
in a hypothetical trajectory is shown.
Adapted from Levine et al. (2012).

Conclusion

This kill switch idea is not yet complete for our project and is still a concept. One issue is that the Phosphorelay gene circuit controlling sporulation initiation is found in B. subtilis, which is a gram positive bacterium and not in E. coli which is a gram negative bacterium. Therefore it is hard to implement this system in E. coli. Another issue is that our plasmids are IPTG induced just like this kill switch, which means that that the timer would start as soon as we introduce IPTG for protein expression.

When implementing the zwitterionic antifouling protein, gene transfer could occur which could be an issue and therefore we thought about an existing kill switch idea that is more probable. The idea is the Gene Guard system (iGEM Imperial College London 2011) which prevents horizontal gene transfer and therefore prevent the exchange of genetic material. A plasmid contains a gene that codes for holin that attaches to the inner membrane and a gene that code for endolysin which causes cell lysis. Antiholin will be integrated in our plasmid and will inhibit holin. This will cause no pores formation in the inner membrane and the endolysin cannot enter the periplasm to break down the outer membrane. This means that the bacteria always need the plasmid with antiholin sequence and the plasmid that encodes for endolysin and holin or else cell lysis occurs in case of plasmid exchange.

Bibliography

[1] D. Huang, W.J. Holtz, M.M Maharbiz. Journal of Biological Engineering. 6:9 (2012). doi: 10.1186/1754-1611-6-9

[2] J.H. Levine, M.E. Fontes, J. Dworkin, M.B. Elowitz. Pulsed Feedback Defers Cellular Differentiation. PLoS biology. 10:e1001252 (2012). doi:10.1371/journal.pbio.1001252

iGEM Team TU Eindhoven 2014

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