In this project our aim is to engineer a biosensor that will be able to detect simultaneously the presence of the nitrite, nitrate and ortophosphate ions in water.

One of the tools that our biosensor uses is de nirK/nsrR system from N.europaea. We can use it too as a new transcriptional regulation system, as a protein coding sequence joined to Pnir (nirK promoter) without the presence of nitrite won't be expressed due to the coupling of NsrR (nitrite sensitive repressor)to Pnir. If we add nitrite to the media, NsrR will be released and the transcription will begin.

For our sensitive bacteria, we will join Pnir to a reporter gene , so we will be able to detect when NO2- concentration reaches a threshold.

To detect the phosphate we will make use of the phoA promoter from E. coli. It's upregulated in the absence of phosphate, so it can be used to detect its presence in waters after coupling it with an inverting system like tetR and then adding a reporter gene.

The nitrate will be detected using narG promoter from E.coli. This promoter takes part in the nitrate reductase system and it has its expression enhanced with the presence of NO3-. We will be able to detect the presence of nitrate coupling the promoter with a reporter protein. We notice it when the concentration of nitrate reaches a threshold.

Nitrates

Nitrogen compounds occur naturally into the soil from decaying plants and animals. Main sources of nitrogen compounds include human sewage and livestock manure; inorganic sources examples are plant fertilizers such potassium nitrate and ammonium nitrate. Many nitrificant bacteria are able to convert the rest of nitrogen compounds to nitrate ions; nitrate is also the form absorbable by plants. However, nitrate is highly leachable so that it can reach and pollute water easily.

The main risk for nitrate is its ability to oxidizes to nitrite, which has the responsibility for the toxic effects (See “nitrites” below). Also, they contribute to increase the cultural eutrophication effect (see “phosphates” below). The Environmental Protection Agency (EPA) has adopted the 10 mg/L standard as the maximum contaminant level (MCL) for nitrate-nitrogen

Nitrites

Nitrite ions have the same primary origin than nitrate ones, owing to the fact that the majority of nitrite ions are produced by the oxidation of nitrate-nitrogen.

It's very important to measure these ions in drinking water, especially for infants, pregnant and nursing women, and elderly people. Nitrite is able to oxidize Fe2+ to Fe3+ and thus transform hemoglobin to methemoglobin. When reaching the first year of life, most people can revert this transformation easily, but there are some risk groups, including babies under 1 year, pregnant and nursing women and elderly people. It is possible for them to get their enzymatic systems saturated and cannot convert methemoglobin to oxyhemoglobin, acquiring methemoglobinemia. This is a pathology in which there isn't hemoglobin enough to satisfy the tissues demands, so that symptoms should be: difficulty to breath and blueness of skin, and in long term diuresis, increased starchy deposits and hemorrhaging of the spleen.

To sum up, it is very important to regulate nitrite concentrations in drinking waters. EPA establish the maximum contaminant level (MCL) for nitrite-nitrogen in about 1 mg/L

Phosphates

Phosphorus allows plants and animals to grow and function, as it is an essential macronutrient. Inside living being, phosphorus is found in its oxidized form, phosphate. It is part of lots and lots of biochemical molecules, such as DNA, proteins, ATP, phospholipids, etc. Thus, phosphates are found in every wastewater and sewage, which constitute the main source of these ions. Also, they can increase their concentration due to the presence of fertilizers and so other farming products which can increase water phosphate levels.

When drinking water with very high levels of phosphates, digestive problems could occur, but they don't have any important toxic effect. The main risk of getting a high concentration of phosphates is produced on the environment; they allow the phenomenon of eutrophication. It is a term for what happens when algae becomes unusually productive on water systems, and is due to the presence in water of a large amount of nutrients. Eutrophication consists of the formation of masses of algae's blooms which choke rivers and lakes.

The consequence of generating such those algae blooms is the massive consumption of resources as oxygen, as well as the cut of light supply. Eutrophicated areas are dead areas, where is impossible for life to develop. The equilibrium of ecosystems is, thus, deeply altered.

Therefore, it is very important to regulate and limit the phosphate concentration in water, even more if this water will end in a reservoir like a lake. EPA establishes the MCL for phosphate in no more than 0,1 mg/L for streams that do not empty into reservoirs, 0.05 mg/L for streams which empty into reservoirs, and 0,025 for reservoirs.

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