Difference between revisions of "Team:Wageningen UR/Description/Biocontainment"

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Because we could image that using …, we wanted to have another approach to achieving biocontainment in parallel, to make BeeT as safe as possible. An idea that came up was using a bacterial strain developed by Mandell and colleagues<sup><a href="#bk1" id="refbk1">1</a></sup> as a chassis for BeeT. This strain can be confined to a certain area because it is auxotrophic for a synthetic amino acid, para-L-biphenylalanine (BipA). Several essential proteins of this bacterial strain were engineered to function only when BipA is incorporated in the active site, leading to death of the bacterium when the synthetic amino acid is not available. In our case, BipA is applied to the beehive, so BeeT cannot survive when it escapes in the environment, or when the beekeepers ceases to supply BipA.
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There is a drawback, however. Even though the GMO dies as soon as it is deprived of the synthetic amino acid, it’s heterologous DNA will remain in the environment. Since DNA is rather stable given the right circumstances<sup><a href="#bk2" id="refbk2">1</a></sup>, it can be transferred to other bacteria via horizontal gene transfer
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<a id="vi1" href= http://www.nature.com/nature/journal/v518/n7537/abs/nature14121.html >1.</a> Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., ... & Church, G. M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518(7537), 55-60.
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<a id="vi1" href= http://aem.asm.org/content/58/9/3012.short >1.</a> Romanowski, G., Lorenz, M. G., Sayler, G., & Wackernagel, W. (1992). Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Applied and Environmental Microbiology,58(9), 3012-3019.
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Revision as of 17:27, 8 October 2016

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Biocontainment

BeeT is intended to be used outside the lab, in beehives, where it is in close contact with nature. We cannot be sure about the effect on existing ecosystems if BeeT would be released in the environment, which is why it is engineered to die if it leaves the beehive. Our first measure to achieve this is a light-induced kill switch that relies on the balance of a bacterial toxin and an antitoxin that are expressed simultaneously. In the dark beehive, the system is unaffected. In the presence of blue light, a component of sunlight, the balance is disturbed in favour of the bacterial toxin. This will kill the bacterium.

As an additional safety measure, BeeT relies on the presence of a synthetic amino acid that is to be applied to the beehive. In the presence of the synthetic amino acid, catalytically dead Cas9 (dCas9) is produced that has the synthetic amino acid incorporated, at the expense of catalytically active Cas9. If BeeT escapes from the hive, the synthetic amino acid is no longer available, and dCas9 can no longer be formed. Instead, catalytically active Cas9 is produced which cuts the BeeT genome as well as any heterologous DNA that is present, thereby killing the bacterium and preventing horizontal gene transfer.

Light Kill Switch

YOUR TEXT HERE

Cas9-based kill switch

Introduction

Because we could image that using …, we wanted to have another approach to achieving biocontainment in parallel, to make BeeT as safe as possible. An idea that came up was using a bacterial strain developed by Mandell and colleagues1 as a chassis for BeeT. This strain can be confined to a certain area because it is auxotrophic for a synthetic amino acid, para-L-biphenylalanine (BipA). Several essential proteins of this bacterial strain were engineered to function only when BipA is incorporated in the active site, leading to death of the bacterium when the synthetic amino acid is not available. In our case, BipA is applied to the beehive, so BeeT cannot survive when it escapes in the environment, or when the beekeepers ceases to supply BipA.
There is a drawback, however. Even though the GMO dies as soon as it is deprived of the synthetic amino acid, it’s heterologous DNA will remain in the environment. Since DNA is rather stable given the right circumstances1, it can be transferred to other bacteria via horizontal gene transfer

References

     

    References for Cas9-based kill switch

    1. Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., ... & Church, G. M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518(7537), 55-60. ↩

    1. Romanowski, G., Lorenz, M. G., Sayler, G., & Wackernagel, W. (1992). Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Applied and Environmental Microbiology,58(9), 3012-3019. ↩