The reason of our project
The plant that we have selected to make our research is Arabidopsis thaliana for so many reasons. For example:
- It is a small plant (around 10-20cm) and it has a very quick life cycle (from 6 weeks since the germination until the production of seeds).
- The genome of Arabidopsis thaliana is completed sequenced.
- It is easy to transform.
For all this reasons we choose to change the reporter protein by the eyGFPuv (a reporter that it could see while it is exposed to UV light) or the caset RUBY (change the colour of the plant into red) In addition, we used PEST sequences in order to degradant quicker the reporter, so when the exposure finishes, the plant would degradant the reporter and the signalling would finish.
How our project works
The promoters
A promoter is a region of the DNA that control the transcription of the gene of a specific region from DNA to RNA. The functioning of the promoters is of so many types and it is very different from one to another. However, there are some big groups in the one we can classify it.
- Constitutive promoters: they induce the transcription of the gene that they refer at whatever is the ambient factors or the location in the plant (tissue, organ...) This makes that transgenic organism that have a constitutive promoters have a huge expression of the gene. In our project we use a constitutive promoter (p35S)
- Inducible promoters: they transcribe the gene once they are induced so the quantity of the protein of interest is fully related with the ambient factors. This ambient factor could be endogenous (hormones) and exogenous (temperature, injures to the plant and the attack of pathogens). In our project we use four constitutive promoters to CO2 (CSY4, ADH1, ATPS, G3PDH)
In our project we use some inducible promoters from the Arabidopsis thaliana which we use them to sense high concentrations of CO2 As a result, we have a plant that reacts at high concentrations of CO2 and show us making a change in its phenotype that can be detected by us. (See “Plant synthetic biology”)
The reporter
A reporter is a sequence of DNA that in synthetic biology has a huge quantity of uses, such us showing that the modification has be made correctly, to show us that the promoter regulation is working correctly, the synthesis of the gene of interest has been result successfully.
In our project we use the reporter to show that the plant has detect a change in the ambient (the inducible promoter has been activated by the substance of interest that we are sensing). As we need to have a plant that show the change in a simple way, we need to make a change in the phenotype of the plant that humans could detect in a simple way (at naked eye, for example).
This is the reason why we selected these two reporters, the caset RUBY and the gene eYGFPuv.
The caset RUBY: is a reporter that transcribes into three enzymes that change the colour of the plant into the characteristic red colour of the beet (B.vulgaris). The caset RUBY is not harmful with the plant because we had grown some A.thaliana whit a constitutive promoter (p35S), and the caset RUBY and they all grow perfectly and at the same time that the wild type A.thaliana. (See “Plant synthetic biology”)
The eYGFPuv gene: Is a gene that comes from the Aequorea victoria and it transcribes into a protein that it shines while the plant is being pointed with UV light. The eYGFP is not harmful with the plant because we had grown some A.thaliana whit a constitutive promoter (p35S), and the eYGFPUV and they all grow perfectly and at the same time, that the wild type A.thaliana.
The degradation sequences
One time the exposure finish, we need that the plant stops the expression of the reporter and that the proteins that had already been created been degradation. The part of not transcribing more reporter is already done (with the inducible promoters), but we still having the reporter in the cell. This is why we add to our construction the PEST sequences.
A PEST sequence is a tail of aminoacid that mark proteins for degradation within the cell, helping to maintain a balance in protein levels int the plant. We use them make a shorter middle life of the proteins. As a result, once the plant is not more exposed to the substance of interest, it did not have more reporter in the cells.
We know that the PEST sequences are not harmful to plants because they are fondued in plants wild type. (See “Plant synthetic biology”)
The future of our project
In the next years, the project would be focused on still developing the construction and making a permanent modification in A.thaliana with the final construction (the inducible promoter, the reporter, the PEST sequence and the terminator). One time we have all these steps done; we should have a completely functional biosensor that one time is exposed to the substance of interest, it would l express the reporter, and one time the exposure finish, the expression will also finish.
In addition, the next teams should start to search new substances of interest that the biosensor will be able to sense (and the inducible promoters that are related with). One of the proposes that we have is that they could detect nitrates that could arrive to the plants, causing the eutrophication of the water and destroying the ecosystem of the water. They could also detect heavy metals that could be harmful at high concentrations, generating a huge quantity of illness (such as problems in the blood, depression, anxiety...) The principal problem is that they could not be degraded at a natural way so by the thopic chain, it could arrive to humans.