Team:UAB-Barcelona/Project2

= Overall project =

Nitrite, nitrate and phosphate
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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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Theoretical basis of the detection of nitrite
We will take advance of the N. europaea nirK system. This system encodes a nitrite reductase that is synthethised in response to high levels of nitrite. The key sequences are:

-the nsrR protein (nitrite sensitive transcriptional repressor), that is constitutive

-the promoter Pnir (that is before the nitrite reductase)

In the absence of nitrite, nsrR is recognising a sequence inside Pnir and is bound blocking the transcription. In the presence of nitrite, it is released and it allows the transcription and synthesis of the downstream protein. In E. coli it works properly too.

So if we couple a reporter protein to Pnir and we add nsrR with a constitutive promoter, we will be able to detect the presence of nitrite. The threshold is around 3 milimolar at pH 5.3

This is our construct:



Theoretical basis of the detection of phosphate
Escherichia coli has a system to produce alkaline phosphatase if there is phosphate starvation.

E. coli detects the low concentration of external phosphate, and after a chain of interactions, PhoB binds to phoA promoter, giving the σ70 binding region the promoter lacks and enhances the transcription and consequently the synthesis of alkaline phosphatase.

Joining phoA to a inverter system (like lacI)and then to a reporter is a mean of detecting phosphate concentration is water



Cloning Pnir and nsrR
We cloned the promoter Pnir and the protein nsrR from N.europaea

First of all, we grew Nitrosomonas europaea. It was a hard work because it grows very slowly. We used the ATCC recommended medium and after a week we could extract the genomic DNA

While growing, we ordered the four primers

We cloned both sequences

A problem we found was the fact that nsrR sequence has a EcoRI target inside. So it wouldn't fulfill the requirements of the standards.As the target is in the very middle of the sequence, we could't introduce a silent mutation with the primers.

The solution would be synthesizing the nsrR sequence with a silent mutation, but we didn't have enough time to order and receive it.

Making new biobricks
Following this cloning strategy we aimed to design this biobricks:

Detection of nitrite

Ptet-RBS-nsr-RBS-GFP-T-T  ---linkerPnir-RBS-tetR-RBS-RFP-T-T  // Biobrick 17

or

pLac-RBS-nsr-RBS-GFP-T-T  ---linkerPnir-RBS-LacI-RBS-RFP-T-T  // Biobrick 18

Using Pnir/nsrR regulation system

Biobrick with Pnir // Biobrick 2

Biobrick with nsrR // Biobrick 3

Cloning phoA
We cloned the promoter phoA from Escherichia coli

First of all we grew E. coli DH5-alpha, and then we extracted the genomic DNA

We ordered the two primers for phoA

After that, we cloned phoA

Making new biobricks
Following this cloning strategy we aimed to design this biobricks:

Detection of phosphate

pLacI-RBS-RFP-T-T  ---linkerPhoa-RBS-LacI-RBS-GFP-T-T // Biobrick 13

or

ptet-RBS-RFP-T-T  ---linkerPhoa-RBS-tetR-RBS-GFP-T-T // Biobrick 14

=Bibliography=

'References'

Nitrite

Beaumont, H.J.E., Lens, S.I., Reijnders, W.N.M., Westerhoff, H.V and van Spanning, R.J.M. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor. Molecular Microbiology (2004) 54: 148–158

Phosphate

Guan, D., Wanner, B. and Inouye, H. Analysis of regulation of phoB expression using a phoB-cat fusion. Journal of bacteriology (Nov 1983) 710-717

Van Dien, S.J. and Keasling, J.K. Analysis of regulation of phoB expression using a phoB-cat fusion. J.theor.biol. (1998) 190, 37-49

Amemura,M., Makino, K., Shinagawa,M., Nakata,A. Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM Is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2. Journal of bacteriology, Nov. 1990, p. 6300-6307

Nitrate

Wang,H. , Tseng, C.P., and .Gunsalus, R.P. The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite. Journal of bacteriology (Sept. 1999,) p. 5303–5308