Team:Toulouse/Project/Spreading

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<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree.  
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<p class="texte">Our engineered bacterium is designed to be inoculated in a tree and to cure fungal diseases. To target the possible environmental issues resulting of using a modified organism directly on trees bordering the Canal du Midi, our team worked on different aspects to ensure a safe use of SubtiTree.  
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria.  
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria.  
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Taking into account these issues, we thought about three modules.
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Taking into account these issues, we imagined three modules.
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<p class="title1">Survival in the environment: proline auxotroph <i>B. subtilis</i></p>
<p class="title1">Survival in the environment: proline auxotroph <i>B. subtilis</i></p>
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<p class="texte">SubtiTree will live in sap tree, thus we use one endophyte <I>Bacillus subtilis</I> strain. In order to contain our bacteria in this area during a short period of time, we modified some of its survival characteristics. To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The proline could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the proline, it will be obliged to take it in its close environment, that is to say the phloem sap.
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<p class="texte">SubtiTree will live in sap tree, thus we will use an endophyte <I>B. subtilis</I> strain. In order to contain our bacteria in this area during a short period of time, we thought of modifying some of its survival characteristics. To make the bacterium growth dependant on the presence of the tree (and therefore avoid spreading in the environment), we planned to use a <i>B.subtilis</i> strain auxotroph for proline. The bacterium should then be unable to synthesize this essential amino acid. Proline is the most abundant amino acid in the phloem sap. If the bacterium is in the sap, it should grow normally without any deficiency, but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time (proline is present only in very low quantities in the ground).  
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Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the proline present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, proline is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/>  
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Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find.</p>
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Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore they should be easy to find.</p> DIRE QUE CE N'EST PAS CE QU'ON A UTILISE??
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p>
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p>
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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\>  
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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 planned to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should die in the fall.<br\>  
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<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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<i>B. subtilis</i> is a sporing bacterium: sporulation enables the microorganism to resist very harsh conditions and to spread from tree to tree.<br/>  
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To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. We chose a <I>B.subtilis</I> strain without late genes of sporulation. Thus, after a season of treatment, the sap become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/>
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To control any unwanted long-term development of SubtiTree, our strain should therefore be deficient for sporulating. We USED or COULD USE a <I>B.subtilis</I> strain deficient in the late genes for sporulation. Thus, during fall, when the sap become less nutritious and the temperature is low, the engineered bacterium will die and not pass through the following winter.<br/>
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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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In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus allowing a better faith on the genetic constructions.
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<p class="texte">These two first characteristics of SubtiTree show that it is an annual bacterium, which can only grow in sap tree. By combining them, they prevent any long term colonization of an ecological niche by SubtiTree against wild type bacteria. These prevent a long term effect.</p>
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<p class="texte">These two first characteristics of SubtiTree demonstrate that it should be an annual bacterium, growing only in sap tree. By combining them, they should prevent any long term colonization of any other ecological niche than the plane trees</p>
<p class="title1">Gene transfer: toxin-antitoxin system</p>
<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, we also wondered about horizontal genes transfer. The goal of this module is to prevent horizontal transfers 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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<p class="texte">We also wondered about horizontal genes transfer. The goal of this module is to prevent horizontal transfers 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 optimized 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/>
<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 optimized 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/>
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/>

Revision as of 15:59, 16 October 2014