Team:Toulouse/Project/Spreading

From 2014.igem.org

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All our constructions are carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta.  
All our constructions are carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta.  
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<p class="texte"><I><CENTER>Survival in the environment</I></CENTER></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. 
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First of all, the control of synthetic bacterium location concerned us,that is why we border it inside the plane tree. Sap tree is known to contain a certain quantity of proline which could allow bacterial growth whereas the soil is composed by only trace of this amino acid. Thus, we decided to delete the proline production pathway to make an auxotrophic bacterium for this amino acid.
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<p class="texte">Secondly, SubtiTree is conceived as an annual bacterium to avoid any kind of mutations. It is known that endophyte bacteria must sporulate to survive to winter. Thus, we chose a <I>B.subtilis</I> strain without late genes of sporulation. So SubtiTree is a non-sporing bacterium, which means that under stress conditions (cold temperature, food lack …) or outside the sap, the bacterium will not resist and die.</p>
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<p class="texte">These two characteristics of SubtiTree show that it is an annual bacterium, which can only grow in sap tree. By combinind 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"><I><CENTER>Gene transfer</I></CENTER></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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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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<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>
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<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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Revision as of 09:54, 13 October 2014