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Team:TU Darmstadt/safety
From 2013.igem.org
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Many different kill switches that are promoters that induced by the presence of an inducer 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. | Many different kill switches that are promoters that induced by the presence of an inducer 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. | ||
<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 doesn’t phosphorylate its cognate response regulator FixJ which then doesn’t drive gene expression from the FixK2 promoter. Downstream of the promoter lies the λ phage repressor cI which represses the strong λ phage promotor pR. | <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 doesn’t phosphorylate its cognate response regulator FixJ which then doesn’t drive gene expression from the FixK2 promoter. Downstream of the promoter lies the λ phage repressor cI 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. | 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. | ||
Revision as of 13:58, 29 September 2013
Why biosafety?
One key issue for the implementation Synthetic Biology in everyday life is safety. Since genetically modified organisms (GMOs) can interact with natural organisms, evolve and adapt to their environment, completely new approaches are needed to address scientific and public safety concerns. Safety measures have to work independently of the operator’s skills or background knowledge.
A light-induced kill switch
Many different kill switches that are promoters that induced by the presence of an inducer 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 doesn’t phosphorylate its cognate response regulator FixJ which then doesn’t drive gene expression from the FixK2 promoter. Downstream of the promoter lies the λ phage repressor cI which represses the strong λ phage promotor pR.
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 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 due to 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 spilling the expression of PezT is induced by the FRET inducing 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 prior autoclaving. All these safety measures raise our device’s applicability and operability in everyday life so our device can be handled theoretically by untrained workers without an increase in risk.
