Team:CIDEB-UANL Mexico/project capture

As a step to desalinize water, iGEM CIDEB 2014's project intended to capture sodium ions from saline water using a protein named NhaS produced from the nhaS gene expression. This gene was obtained from a 1994 patent by Krulwich & Ivey and no further information was found about the gene in other sources. A lot of research of related information and predictions about the NhaS protein in different sites had to be made in order to obtain more information about it and being able to work with and include it in the project.

Description

NhaS is a putative protein from Bacillus firmus that is characterized by its ability to bind and sequestering sodium ions, with a calculated weight of 7100 Daltons and a pH of 12. It “can enhance the Na⁺ -resistance of antiporter- deficient strains by increasing the availability of Na⁺ to the integral membrane antiporters on the cytoplasmic side of the membrane and by sequestering Na⁺ while rate-limiting efflux mechanisms catalyze extrusion of the cation.” (Krulwich & Ivey, 1994)

Research by Krulwich and Ivey (1994) supports that in its origin bacteria, NhaS works as a regulatory of pH in protein's homeostasis because it makes the cytoplasmic pH more acidic than the external medium, usually basic.

Essentially, NhaS performs three different functions; (1) capturing sodium ions, (2) regulating pH, and (3) enhancing the resistance of bacteria to high saline conditions.

What would happen with Cl- ions?

After the removal of sodium ions from salty water, it is needed to remove Cl- ions as well in order to complete the desalination process. E. CARU only captures Na+ ions, leaving Cl- ions in the water medium; but since Cl is a diatomic molecule (meaning it cannot be alone in normal conditions), it joins another Cl molecule, forming Cl₂.

Normally, Cl₂ is in a gaseous state at normal conditions, so what would remain after E. CARU takes Na+ ions from water is a mixture of Cl₂ gas and water molecules. In order to remove it from water it is possible to use a method involving the separation of a gas from a liquid based in the boiling points of each component in the mixture. Cl₂ gas has a boiling point of -34.6°C and water has a boiling point of 100°C. By cooling the mixture at -34.6°C, the gas would evaporate, separating itself from water. (Bentor, 2014).

Yet still, there is an important factor to consider: Cl₂ gas is toxic, but as it is 2.5 times heavier than air (CFC StarTec LLC, 2007), it would stay in water at room temperature. For this reason, before cooling the Cl₂ gas in order to take it away, it is necessary to take safety measurements. The one proposed by the team is the usage of an Atomic Absorption Spectroscopy (see Figure 2).

Atomic Absorption Spectrometry (AAS) is an analytical technique that measures the concentrations of elements. Atomic absorption is so sensitive that it can measure down to parts per billion of a gram (µg dm–3). The technique uses light wavelengths specifically absorbed by an element that correspond to the energies needed to excite electrons from one energy level to a higher one. (Royal Society of Chemistry).

Figure 2. Atomic Absorption Spectroscopy dispositive.

The use of an Atomic Absorption Spectrometry is a way to remove the Cl₂ gas from water and measure it to prevent its escape. Then, the stored Cl₂ gas can be used to sterilize drinking water, to disinfect swimming pools and to be part in the manufacture of many consumer products; such as paper, dyestuffs, textiles, petroleum products, medicines, antiseptics, insecticides, foodstuffs, solvents, paints, and plastics (especially PVC). It can also be used to produce bleaches, chlorates, chloroform, carbon tetrachloride and bromine. A further substantial use for this element is in organic chemistry, both as an oxidizing agent and in substitution reactions (Emsley, 2011).

Research on NhaS

It is important to be familiarized with what it is being worked with, and since this putative protein has never been used at iGEM before, it was done a lot of research on it.

The composition and form of a protein show relevant data about its actions and functions, that it is why it was investigated NhaS’ predicted type. It was found in the modelling tool 3D-JIGSAW from Cancer Research UK' site that its possible protein or peptide type would be helix, coil or strand (Figure 3). Also, it was found in the Raptor X protein modelling site a predicted structure of the protein. (Figure 4)

Research was done in different sources. The previous information was confirmed in Predict Protein site. As shown in Figure 5 and Figure 6.

As it can be apprecciated, the results from all sources match, so, based on the previous information it was concluded that NhaS is most possible to be of the helix type. Being aware of the secondary structure of proteins is relevant, since hence, the protein folding mechanism can be taken into consideration.

According to Krulwich and Ivey (1994), the location of the protein is in the cytoplasmic side of the membrane, however, when the research was made, it was found out that the protein is predicted to be highly non-cytoplasmic (Stockholm Bioinformatics Center SBC Phobius, 2014), as shown in Figure 7.

This makes an antithesis of theories. Also as it is presented in Figure 5, NhaS is predicted to be exposed and buried, which accords with Figure 6, that show how the amino acids sequence of Nhas is divided many times in exposed and hidden. As nobody has described exactly how is NhaS and where it would be placed inside E. coli, the team, based on all the modeling above, came out with a hypothesis in which NhaS would cross two times the membrane as it is shown in the Figure 7, having two parts exposed, the beginning loop and the final helix with loop, and an inner part, consisting in the two big helixes as transmembrane and the little loop in the middle of the helixes in the cytoplasmic side. The determinant factor was the structure of NhaS predicted by Raptor X (Figure 4) that is very similar to the transmembrane proteins and ion channels, which are in similar positions, as NhaS in our hypothesis, inside the bacteria.

This prediction was used for the understanding and explaining of the module, as well as for designing different animations.

Further information about NhaS and the experimental results made by the team about it can be found in its parts registry section, as well as the results section in this wiki.

Device

The proposed circuit for the module is as follows: An UV promoter, an RBS; the nhaS gene, another RBS, a WinterGreen odor reporter (BSMT1 Opt.) and a terminator.

However, for practical and experimental reasons explained in the next paragraph, it was decided that nhaS and BSMT1 Opt. would be tested separately, leading to both Capture and Aroma modules.

As mentioned before, in the ideal project it is proposed an odor Wintergreen (BSMT1 Opt.) reporter for this module in order to know if there is any production and expression of NhaS, but since BSMT1 Opt. wasn't proved yet and NhaS is a putative protein it was decided to test NhaS physically with RFP, which is a simple and common reporter, in order to observe functional results in the tests for this specific segment of the project, obtaining conclusions about both genes, and being able to put them together in the ideal device.

Further information about the odor reporter can be found in the Aroma module in this wiki.

Capture module's parts description

Justifications

UV promoter as an initiator for the system

Other teams that used pUV and NhaS

In the case of pUV

Before choosing the BBa_I765001 UV promoter the team reviewed the experiences by other participants with it, with the intention of assuring the maximum possible success rate if used in this module. The following table shows information about its usage with other teams.

In the case of nhaS

There are no teams that used this gene in the past. IGEM CIDEB 2014 was the first team that synthesized, characterized, tested and registered it in the parts registry.

Future usage

The team considers NhaS to be useful for the scientific community in general, in addition to iGEM. NhaS, as mentioned before, is a putative protein. Knowing specific results about its functions could be of great use for future projects.

The potential for future usage of this gene is great. As mentioned in the patent from Krulwich and Ivey (1994), the gene encoding NhaS can be introduced into cells to produce desalination bioreactors, can be introduced into plants as a transgene to produce plants that are resistant to sodium, may be used for treatments involving NNa₊ /K₊ ATPase disorders, e.g., in heart disease, and may be introduced parenterally, preferably orally, to bind to and sequester dietary sodium. The possibilities are wide and promising.

Capture Module Zoom In

Bibliography/References