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Team:Evry/Project/Compounds/Sensing
From 2014.igem.org
The sensing approach
Advantages of bio-sensing
In response to this recent awareness towards those toxic compounds, different systems of detection have been developed.
However with the approach of bio-sensing, we develop tools which are able to detect a pollutant with a very high efficiency, and with a great specificity.
Biological elements necessary to build those tools are cheap, easily obtained and their production doesn’t emit any pollutant.
Thus besides being effective and cheap systems, biosensors are totally biological and non-polluting tools.
Systems
To develop our bio-sensors, we looked for some natural systems based promoters inducibles by our compounds of interest. It is often promoters wich allow the expression of a set of genes which correspond to the cell's response to the compound.
For phenols, a set of genes called Dmp operon in Pseudomonas CF600 is able to degrade phenol to produce acetyl CoA, and use this molecule as an energy source.
Figure6: The catabolic pathway for degradation of phenol and the organization of Dmp operon. (Powlowski J, Shingler V., 1994)
The transcription of this operon is regulated by the DmpR regulator element which binds phenol and activates the transcription of the phenol hydroxylase enzyme by allowing the fixation of the RNA polymerase (more information, see the section Sensors).
For PCBs, two distinct classes of bacteria have now been identified as being able to degrade PCBs:
- Aerobic bacteria which live in oxygenated environments
- Anaerobic bacteria which live in oxygen free environments such as aquatic sediments.
Futur : degradation?
Sensing constructions are very simple and easy to construct. In a larger framework we can imagine one day being able to clone all the enzymes of the degradation pathways of phenol and PCBs in our bacteria. In fact we can create more than a sensor system based on sponge: a complete filtrating system which can not only sense pollutants, but totally remove them.
For removing nitrites, it exists different biological denitrification systems to reduce nitrates concentration in water that use bacteria as Pseudomonas with a denitrification yield of 80%. Bacteria are fixed on a mineral support and feed with acetic acid or ethanol (SNIDE). The major drawback is the production of nitrous and nitric oxide that are greenhouse gases.
To make a bacterium able to transform nitrites into nitrogen we just have to add two enzymes:
-> Hydroxylamine oxydase from Parococcus denitrificans: nitrite + H2O = hydroxylamine
-> Hydrazine oxydoreductase from Candidatus Brocadia anammoxidans: hydroxylamine + NH3 + acceptor = N2 + H2O + reduced acceptor
Figure8: Nitrogen cycle.
Unfortunately, dealing with heavy metals means dealing with atoms which cannot just be degraded by a biological pathway. But we can imagine some ways of accumulation of these elements in bacteria that we can remove and treated after like chemical waste.
They use different mechanisms, aerobes attack PCBs oxidatively, breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle.
Figure7: The catabolic pathway for degradation of biphenyl by aerobic bacteria and the organization of the bph gene cluster (Kensuke F., Hidehiko F., 2008) .
The transcription of this set of genes is regulated by bphR2 which binds PCBs and activates the transcription of pbhR1 gene (more information, see the section Sensors).
For nitrites, the degradation pathways are very well known because they belong to the nitrite cycle. As for heavy metals, a lot of operons which allows the cell tolerance to these compounds exist and have promoters reacting specifically to the metal concentration.
However for this project, we chose to develop systems based on promoters presents in our bacteria strain Pseudovibrio denitrificans.
In this way, we used an RNA sequencing approach to detect promoters able to react to the pollutant presence (more information, see the section RNAseq).
Our bacteria strain is known to live in sponge and may provide them a better tolerance to pollutants like heavy metals. The ability of the bacteria is currently study by the 'Molecules of defence and communication in the microbial ecosystems' team of the National museum of natural history of Paris (their web page here)
