Team:Marburg:Safety

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SURFkiller
SURFkiller
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The work with genetically modified organisms (GMOs) harbours the risk of their release to the nature. After release, serious consequences such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the environment or to make their survival in nature impossible. For this purpose, we decided to develop a security system for Bacillus subtilis. This system is based on one of the cells most important compartment- the ribosome. Ribosomes are essential as they are responsible for the protein synthesis and a loss of them leads to a halt of protein biosynthesis resulting in a rapid cell death.
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<div class="figure" style="float: right; width: 25%; margin-bottom: 10px; min-width: 25%; margin-left: 15px; margin-top: 28px;">
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<a href="https://static.igem.org/mediawiki/2014/f/fa/IGEM_Marburg_2014_-_The_SURFkiller.mp4">
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<img src="https://static.igem.org/mediawiki/2014/8/8e/MR_slider_surfkiller.jpg" alt="SURFkiller" style="width:100%;"/>
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</a>
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<span class="caption">
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See our short video description on <a href="https://www.youtube.com/watch?v=vKbsz4ffzYY">YouTube</a>
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or <a href="https://static.igem.org/mediawiki/2014/f/fa/IGEM_Marburg_2014_-_The_SURFkiller.mp4">download it</a>.</span>
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</div>
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</p><p>
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The work with genetically modified organisms (GMOs) harbours the risk of their release into nature. After release, serious consequences  
 +
such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the  
 +
environment or to make their survival in nature impossible. For this purpose, we decided to develop a security system for <i>Bacillus subtilis</i>.  
 +
This system is based on one of the cells most important compartment- the ribosome. Ribosomes are essential as they are responsible for the  
 +
protein synthesis and a loss of them leads to a halt of protein biosynthesis resulting in a rapid cell death.
 +
</p><p>
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To minimize the risk of failure of the SURFkiller as well as to ensure maximum safety we designed a system composed out of three interconnected
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modules which enable the survival of cells under laboratory conditions but not in the environment. All genes coding for components of our
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SURFkiller were deleted in the organism in order to guarantee a tight control of our system. Before deleting the genes the modules presented
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in figure 1 were inserted via homologous recombination into the genome. First of all module two was integrated into the <i>amyE</i> locus.
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Afterwards module two was placed into the <i>lacA</i> locus of <i>B. subtilis</i>, followed by the insertion of both, module two and three into
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the <i>amyE</i> locus.
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</p>
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</html>
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To minimize the risk of failure of the SURF Killer as well as to ensure maximum safety we designed a system composed out of three interconnected modules which enable the survival of cells under laboratory conditions but not in the environment. All genes coding for components of our SURF Killer were deleted in the organism in order to guarantee a tight control of our system. Before deleting the genes the modules presented in figure 1 were inserted via homologous recombination into the genome. First of all module 2 was integrated into the amyE locus. Afterwards module one and two was placed into the lacA locus of Bacillus subtilis.
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Laboratory conditions and the environment are distinguished by the presence of IPTG (Isopropyl-β-D-thiogalactopyranosid), which is an artificial substrate for the LacI repressor. This compound is added to the medium in the laboratory but is absent in nature, leading to the activation of the SURFkiller system and cell death.  
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Laboratory conditions and the environment are distinguished by the presence of IPTG (Isopropyl-β-D-thiogalactopyranosid), which is an artificial substrate for the LacI repressor. This compound is added to the medium in the laboratory but is absent in nature, leading to the activation of the SURF Killer system and cell death.  
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This cell death is induced by the lack of the essential ribosomal L5 protein. This protein is essential for the assembly of the ribosomal subunits. In the absence of L5 the small and large subunits are synthesized but are not able to form functional ribosomes.  
This cell death is induced by the lack of the essential ribosomal L5 protein. This protein is essential for the assembly of the ribosomal subunits. In the absence of L5 the small and large subunits are synthesized but are not able to form functional ribosomes.  
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<html><div style="margin:0 auto;"><img src="https://static.igem.org/mediawiki/2014/7/7b/MR_killswitch_1.png" alt="scheme" width="55%" style="margin: 0 auto;"><br /><span class="caption">Fig. 1: Scheme of the SURF Killer system.<br />
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<html><div class="figure" style="width:65%;">
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<img src="https://static.igem.org/mediawiki/2014/7/7b/MR_killswitch_1.png" alt="scheme" />
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<span class="caption"><b>Figure 1: Scheme of the SURFkiller system.</b><br />
T4-Holin: Toxin -> lysis<br />
T4-Holin: Toxin -> lysis<br />
T4-Antiholin: Antitoxin -> prevents lysis by Holin<br />
T4-Antiholin: Antitoxin -> prevents lysis by Holin<br />
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L5: essential ribosomal protein</span></div></html>
L5: essential ribosomal protein</span></div></html>
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YvyD is affecting the ribosomes as well. In comparison to L5, YvyD is influencing already assembled ribosomes and inactivates them by dimerization. Those dimers are no longer capable of protein biosynthesis (Lauber et al., 2009). The expression of yvyD (module three) is under the control of a constitutive promoter, regulated by the TetR repressor which is produced by module 1.
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YvyD is affecting the ribosomes as well. In comparison to L5, YvyD is influencing already assembled ribosomes and inactivates them by dimerization. Those dimers are no longer capable of protein biosynthesis (Lauber et al., 2009). The expression of <i>yvyD</i> (module three) is under the control of a constitutive promoter, regulated by the TetR repressor which is produced by module one.
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Additional security is provided by synthesis of the antagonising toxin (T4-Holin; BBa_K112000) and antitoxin (T4-Antiholin). T4-Holin from the T4 phage is lysing the cells by introducing holes into the membrane. However T4-Antiholin, also from the T4 phage, is preventing this by plugging the holes (Wang et al., 2000; Young and Bläsi, 1995). If a mutation occurs in module 2, the T4-Holin will immediately lyse the cells.  
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Additional security is provided by synthesis of the antagonising toxin (<html><a href="http://parts.igem.org/Part:BBa_K112000" target="_blank">T4-Holin</a></html>) and antitoxin (T4-Antiholin). T4-Holin from the T4 phage is lysing the cells by introducing holes into the membrane. However T4-Antiholin, also from the T4 phage, is preventing this by plugging the holes (Wang et al., 2000; Young and Bläsi, 1995). If a mutation occurs in module two, the T4-Holin will immediately lyse the cells.  
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<html><img src="https://static.igem.org/mediawiki/2014/2/27/MR_killswitch_holin-antiholin.png" alt="scheme" width="50%" style="margin: 0 auto;"><img src="https://static.igem.org/mediawiki/2014/8/8c/Holin-lysis.png" alt="scheme" width="50%" style="margin: 0 auto;"><span class="caption"><br />Figure 2: Effectiveness of Holin and Antiholin</span></html>
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<html><div class="figure" style="width:70%;" >
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<img src="https://static.igem.org/mediawiki/2014/0/0d/MR_holin_antiholin.png" alt="scheme"/>
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<div style="clear:both;"></div>
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<span class="caption"><br /><b>Figure 2: Effectiveness of Holin and Antiholin.</b></span>
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</div></html>
The major regulator IPTG is inactivating the LacI repressor resulting in the constant activation of module one and two. In the case of an escape of the modified cells in nature, LacI is no longer inactivated and represses module one and two. This inactivation leads finally to cell death (Figure 3).
The major regulator IPTG is inactivating the LacI repressor resulting in the constant activation of module one and two. In the case of an escape of the modified cells in nature, LacI is no longer inactivated and represses module one and two. This inactivation leads finally to cell death (Figure 3).
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<html><div style="margin: 0 auto;"><img src="https://static.igem.org/mediawiki/2014/9/9f/MR_killswitch_lab.png" alt="scheme" width="50%" style="margin: 0 auto;"><img src="https://static.igem.org/mediawiki/2014/6/64/MR_killswitch_nature.png" alt="scheme" width="50%" style="margin: 0 auto;" /><span class="caption">Figure 3: The SURFkiller in the lab and in nature</span></div></html>
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<html><div class="figure_two" style="width: 100%;">
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<img src="https://static.igem.org/mediawiki/2014/9/9f/MR_killswitch_lab.png" alt="scheme" style="width:48%;float:left;" />
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<html><hr /></html>
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<img src="https://static.igem.org/mediawiki/2014/6/64/MR_killswitch_nature.png" alt="scheme" style="width:48%;float:right;" />
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<div style="clear:both;"></div>
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<span class="caption"><b>Figure 3: The SURFkiller in the lab and in nature.</b></span>
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</div>
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<hr /></html>
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Wang I., Smith DL., Young R., (2000). HOLINS: The Protein Clocks of Bacteriophage Infections. Annu. Rev. Microbiol. 2000. 54:799–825.
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Lauber, M.A., Running, W.E., and Reilly, J.P. (2009) B. subtilis ribosomal proteins: structural homology and post-translational modifications. J Proteome Res 8: 4193–206 http://www.ncbi.nlm.nih.gov/pubmed/19653700. Accessed October 6, 2014.
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Young R., Bläsi U., (1995). Holins: forms and function in bacteriophage lysis. FEMS Microbiology Reviews 17 (1995) 191-205.
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Wang, I.N., Smith, D.L., and Young, R. (2000) Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54: 799–825 http://www.ncbi.nlm.nih.gov/pubmed/11018145. Accessed October 13, 2014.
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Lauber MA., Running WE., Reilly JP., (2009). B. subtilis Ribosomal Proteins: Structural Homology and Post-Translational Modifications. J. Proteome Res. 8 (2009) 4193-4206.
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Young, R., and Bläsi, U. (1995) Holins: form and function in bacteriophage lysis. FEMS Microbiol Rev 17: 191–205 http://www.ncbi.nlm.nih.gov/pubmed/7669346. Accessed October 14, 2014.  
{{Team:Marburg/Template:End}}
{{Team:Marburg/Template:End}}

Latest revision as of 03:36, 18 October 2014

SURFkiller

SURFkiller See our short video description on YouTube or download it.

The work with genetically modified organisms (GMOs) harbours the risk of their release into nature. After release, serious consequences such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the environment or to make their survival in nature impossible. For this purpose, we decided to develop a security system for Bacillus subtilis. This system is based on one of the cells most important compartment- the ribosome. Ribosomes are essential as they are responsible for the protein synthesis and a loss of them leads to a halt of protein biosynthesis resulting in a rapid cell death.

To minimize the risk of failure of the SURFkiller as well as to ensure maximum safety we designed a system composed out of three interconnected modules which enable the survival of cells under laboratory conditions but not in the environment. All genes coding for components of our SURFkiller were deleted in the organism in order to guarantee a tight control of our system. Before deleting the genes the modules presented in figure 1 were inserted via homologous recombination into the genome. First of all module two was integrated into the amyE locus. Afterwards module two was placed into the lacA locus of B. subtilis, followed by the insertion of both, module two and three into the amyE locus.

Laboratory conditions and the environment are distinguished by the presence of IPTG (Isopropyl-β-D-thiogalactopyranosid), which is an artificial substrate for the LacI repressor. This compound is added to the medium in the laboratory but is absent in nature, leading to the activation of the SURFkiller system and cell death. This cell death is induced by the lack of the essential ribosomal L5 protein. This protein is essential for the assembly of the ribosomal subunits. In the absence of L5 the small and large subunits are synthesized but are not able to form functional ribosomes.

scheme Figure 1: Scheme of the SURFkiller system.
T4-Holin: Toxin -> lysis
T4-Antiholin: Antitoxin -> prevents lysis by Holin
YvyD: Hibernation factor like -> ribosomes not functional
L5: essential ribosomal protein

YvyD is affecting the ribosomes as well. In comparison to L5, YvyD is influencing already assembled ribosomes and inactivates them by dimerization. Those dimers are no longer capable of protein biosynthesis (Lauber et al., 2009). The expression of yvyD (module three) is under the control of a constitutive promoter, regulated by the TetR repressor which is produced by module one. Additional security is provided by synthesis of the antagonising toxin (T4-Holin) and antitoxin (T4-Antiholin). T4-Holin from the T4 phage is lysing the cells by introducing holes into the membrane. However T4-Antiholin, also from the T4 phage, is preventing this by plugging the holes (Wang et al., 2000; Young and Bläsi, 1995). If a mutation occurs in module two, the T4-Holin will immediately lyse the cells.

scheme

Figure 2: Effectiveness of Holin and Antiholin.

The major regulator IPTG is inactivating the LacI repressor resulting in the constant activation of module one and two. In the case of an escape of the modified cells in nature, LacI is no longer inactivated and represses module one and two. This inactivation leads finally to cell death (Figure 3).

scheme scheme
Figure 3: The SURFkiller in the lab and in nature.

Lauber, M.A., Running, W.E., and Reilly, J.P. (2009) B. subtilis ribosomal proteins: structural homology and post-translational modifications. J Proteome Res 8: 4193–206 http://www.ncbi.nlm.nih.gov/pubmed/19653700. Accessed October 6, 2014.

Wang, I.N., Smith, D.L., and Young, R. (2000) Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54: 799–825 http://www.ncbi.nlm.nih.gov/pubmed/11018145. Accessed October 13, 2014.

Young, R., and Bläsi, U. (1995) Holins: form and function in bacteriophage lysis. FEMS Microbiol Rev 17: 191–205 http://www.ncbi.nlm.nih.gov/pubmed/7669346. Accessed October 14, 2014.