Team:Toulouse/Project/Spreading

From 2014.igem.org

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It is known that endophyte bacteria must sporulate to survive to winter. In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\>  
It is known that endophyte bacteria must sporulate to survive to winter. In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\>  
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/>  
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/>  
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To keep the control on the development of SubtiTree, our strain should therefore be non-sporinghus. We chose a <I>B.subtilis</I> strain without late genes of sporulation. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/>
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To keep the control on the development of SubtiTree, our strain should therefore be non-sporingh. We chose a <I>B.subtilis</I> strain without late genes of sporulation. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/>
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.
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<p class="title1">Gene transfer</p>
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<p class="title1">Gene transfer : toxin-antitoxin system</p>
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<p class="texte"> While we were trying to respond to bacterial spreading problem, at the same time we wondered about horizontal gene transfer. The transmission of genes from our synthetic bacterium to wild type bacteria could be blocked with two modifications.
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<p class="texte"> While we were trying to respond to bacterial spreading problem, we also wondered about horizontal gene transfer.The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.
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First, as used in genetic engineering, plasmids are a tremendous way to transfer some genic information. In this way, it is not the best way to restrict the collecting transgenic DNA. That is why we inserted our genes in the bacterium chromosome.  
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<br>Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example <i>tse2</i>) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/>
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Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/>
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To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless).  
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<p class="texte">Secondly, some bacteria are able to accept chromosomal DNA, like <I>Bacillus subtilis</I>. Thus, the previous strategy is  not efficient enough to prevent transfer. We could include the toxin/anti-toxin system. The toxin gene is near our synthetic gene, contrary to the anti-toxin gene. So if there is a transfer of our synthetic gene, the toxin gene will be included in the transfer. Receiver bacteria will die by the production of the toxic protein. </p>
 
<p class="texte">Our synthetic genes are not the only problem in the design of SubtiTree. One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction of SubtiTree in the environment. It is possible to delete this resistance in chromosome. To conclude, the spreading limitation shown previously makes the use of SubtiTree acceptable in the environment.  
<p class="texte">Our synthetic genes are not the only problem in the design of SubtiTree. One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction of SubtiTree in the environment. It is possible to delete this resistance in chromosome. To conclude, the spreading limitation shown previously makes the use of SubtiTree acceptable in the environment.  
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</p>
 
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<p class="title1">Using a toxin-antitoxin system</p>
 
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<p class="texte">
 
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The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/>
 
-
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example <i>tse2</i>) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/>
 
-
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/>
 
-
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless).
 
</p>
</p>

Revision as of 10:38, 13 October 2014