Team:TU Darmstadt/safety

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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/solution">
 
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Why biosafety?</font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular">Why biosafety?</font></h2>
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Many different kill switches that are promoters which are induced by the presence of a signal like IPTG or heat. Since these kill switches can malfunction due to human failure we chosed a different approach that fits perfectly to our device: A kill switch that is simply induced by blue light.
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Many kill switches include promoters which are induced by the presence of a signal like IPTG or heat. Since these kill switches can malfunction due to human failure, we chose a different approach that fits perfectly to our device: A kill switch that is induced by blue light.
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<i>Ohlendorf et al.</i><sup>[1]</sup> constructed the pDawn vector which contains the blue light sensitive histidine kinase YF1. In the presence of blue light YF1 does not phosphorylate its cognate response regulator FixJ which then does not drive gene expression from the FixK2 promoter. Downstream of the promoter the λ phage repressor cI is located which represses the strong λ phage promotor pR.  
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<i>Ohlendorf et al.</i><sup><span style="color:blue"> <font color="#F0F8FF" face="Arial regular">[1]</font></span></sup> constructed the pDawn vector which contains the blue light sensitive histidine kinase YF1. In the presence of blue light YF1 does not phosphorylate its cognate response regulator FixJ which then does not drive gene expression from the FixK2 promoter. Downstream of the promoter the λ phage repressor cI is located which represses the strong λ phage promotor pR.  
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Downstream of this promotor lies our toxin of choice, PezT, which was characterized by <i>Mutschler et al.</i><sup>[2]</sup> to be a very strong inhibitor of cell growth. PezT is a kinase that phosphorylates uridine diphosphate-<i>N</i>-acetylglucosamine (UNAG) and part of a pneumococcal toxin-antitoxin system which induces cell death under stress conditions. UNAG is an essential precursor in the peptidoglycan biosynthesis and phosphorylation of UNAG inhibits in <i>E. coli</i> cell wall synthesis in such an effective manner that studies failed in the past only due to its toxicity.
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Downstream of this promotor lies our toxin of choice, PezT, which was characterized by <i>Mutschler et al.</i><sup><span style="color:blue"> <font color="#F0F8FF" face="Arial regular">[2]</font></span></sup> to be a very strong inhibitor of cell growth. PezT is a kinase that phosphorylates uridine diphosphate-<i>N</i>-acetylglucosamine (UNAG) and is part of a pneumococcal toxin-antitoxin system which induces cell death under stress conditions. UNAG is an essential precursor in the peptidoglycan biosynthesis and phosphorylation of UNAG inhibits in <i>E. coli</i> cell wall synthesis in such an effective manner that studies failed in the past only because of its toxicity.
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In the absence of blue light (or day light respectively) the PezT toxin is not expressed and the bacteria are alive. After the FRET measurement, spilling of PezT is induced by the FRET blue light or day light. Even if the spill remains unbeknownst to the operator, the expression of the toxin will be induced by day light and the leaking bacteria will contain themselves. Used capsules can be disposed without previous autoclaving. All these safety measurements raise our device’s applicability and operability in everyday life so that our device can be handled theoretically by untrained workers without any increase of risk.
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In the absence of blue light (or day light respectively) the PezT toxin is not expressed and the bacteria are alive. After the FRET measurement or a spilling the expression of PezT is induced by the blue light used in the FRET measurement or day light. Even if the spill remains unbeknownst to the operator, the expression of the toxin will be induced by day light and the leaking bacteria will contain themselves. Used capsules can be disposed without previous autoclaving. All these safety measurements raise our device’s applicability and operability in everyday life so that our device can be handled theoretically by untrained workers without any increase of risk.
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<a href="https://2013.igem.org/Team:TU_Darmstadt/safety/Labjournal">
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<img alt="Labbook" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif">
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<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book</font><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Safety form</font></h2>
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<a href="https://static.igem.org/mediawiki/2013/9/9e/TU_Darmstadt_IGEM_2013_Basic_Safety_Form.pdf">
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<font size="3" color="#F0F8FF" face="Arial regular"><b>Safety form were approved on October 3, 2013 by Evan Appleton.<b></font>
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<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2>
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<img alt="Labbook" border="5" src="/wiki/images/8/8f/Book3.jpg">
 
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Latest revision as of 03:48, 5 October 2013







Why biosafety?

safety One key issue for the implementation of Synthetic Biology in everyday life is safety. Since genetically modified organisms (GMOs) can interact with natural organisms as well as evolve and adapt to their environment, completely new approaches are needed to address scientific and public safety concerns. Safety measurements have to work independently from the operator’s skills or background knowledge.


A light-induced kill switch

Many kill switches include promoters which are induced by the presence of a signal like IPTG or heat. Since these kill switches can malfunction due to human failure, we chose a different approach that fits perfectly to our device: A kill switch that is induced by blue light. Ohlendorf et al. [1] constructed the pDawn vector which contains the blue light sensitive histidine kinase YF1. In the presence of blue light YF1 does not phosphorylate its cognate response regulator FixJ which then does not drive gene expression from the FixK2 promoter. Downstream of the promoter the λ phage repressor cI is located which represses the strong λ phage promotor pR.
safety
Downstream of this promotor lies our toxin of choice, PezT, which was characterized by Mutschler et al. [2] to be a very strong inhibitor of cell growth. PezT is a kinase that phosphorylates uridine diphosphate-N-acetylglucosamine (UNAG) and is part of a pneumococcal toxin-antitoxin system which induces cell death under stress conditions. UNAG is an essential precursor in the peptidoglycan biosynthesis and phosphorylation of UNAG inhibits in E. coli cell wall synthesis in such an effective manner that studies failed in the past only because of its toxicity.

What happens after a spill?

In the absence of blue light (or day light respectively) the PezT toxin is not expressed and the bacteria are alive. After the FRET measurement or a spilling the expression of PezT is induced by the blue light used in the FRET measurement or day light. Even if the spill remains unbeknownst to the operator, the expression of the toxin will be induced by day light and the leaking bacteria will contain themselves. Used capsules can be disposed without previous autoclaving. All these safety measurements raise our device’s applicability and operability in everyday life so that our device can be handled theoretically by untrained workers without any increase of risk.




Labbook Visit our lab book



Safety form


Safety form were approved on October 3, 2013 by Evan Appleton.

References

  1. Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K., and Möglich, A. (2012) From dusk till dawn: one-plasmid systems for light-regulated gene expression. J. Mol. Biol. 416, 534–42
  2. Mutschler, H., Gebhardt, M., Shoeman, R. L., and Meinhart, A. (2011) A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis. PLoS Biol. 9, e1001033

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