Team:CIDEB-UANL Mexico/project capture

In order to desalinize water our project intends to capture sodium ions from saline water using a protein produced from the NhaS gene expression. NhaS is a putative protein from Bacillus firmus that is characterized by its ability to bind and sequestering sodium ions. 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, 1992)

Research by Krulwich and Ivey (1992) supports that in its origin bacteria, NhaS works as a regulation pH homeostasis protein because it makes the cytoplasmic pH more acidic than the external medium, usually basic. The calculated weight of the protein is 7100 Daltons and the final protein product is very basic with a calculated pH of 12.

Basically, NhaS enhance the resistance of bacteria to high saline conditions, regulates pH and captures sodium ions.

Figure 1: Patent US 5346815 A shows extracts of the E. coli EP432 transformed with pGEM (fig. 4A) and pGRVH (fig. 4B). The first one is a control plasmid and the second a plasmid with the NhaS gene. Those are crude extracts that were shown by the effect of putting the bacteria to an SFBI excitation, which is a sodium-sensitive molecule used to measure intracellular Na+. Resuming, it shows in basic draws that the protein is expressed in E. coli and in what quantity according to the excitation level where it is exposed.

Research on NhaS

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

The composition and form of a protein show relevant data about its actions and functions, that is why we investigated NhaS’ predicted type. We found in the modelling tool 3D-JIGSAW from Cancer Research UK's site that its possible protein or peptide type would be helix, coil or strand.

Figure 2: Interactive 3D-Jigsaw's result that indicates the predicted protein type of NhaS.

The previous information was confirmed in Predict Protein site.

Figure 3: Results given by Predict Protein showing the secondary structure composition and solvent accessibility of the putative NhaS gene.

Figure 4: Results given by Predict Protein showing the predicted precise structure of the NhaS protein.

Based on the previous information we conclude 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 (1992), the location of the protein is in the cytoplasmic side of the membrane, however, when we made our research, we found out that the protein is predicted to be highly non-cytoplasmic (Yeast Resource Center, 2014). We came out with a hypothesis in which NhaS would be located in the inner part of the membrane but on its cytoplasmic side. This would explain both predictions of both sources of information.

Figure 5: Predicted protein overview results from Yeast Resource Center.

As a “confirmation” for our hypothesis, Predict Protein site gave us the following result:

Figure 6: “Predicted localization for the Bacteria domain: Inner Membrane (GO term ID: 0005886) Prediction confidence 76.”

Based on this predicted result and with the previous hypothesis we formulated, the team concluded that the protein would act in the cytoplasmic side of the inner membrane. This information was used for the understanding and explaining of the module, as well as for designing different animations.

Further information about NhaS can be found in its parts registry section.

How is NhaS gene activated?

In our project we are going to activate NhaS’ production and functionability using a promoter from iGEM Colombian Team 2007 that is regulated by UV irradiation and does not causes mutations in the bacteria (iGEM Colombian Team, 2007). We decided to use this type of promoter in order to have a control about the NhaS action in E. CARU, our project. With this, we can decide at which time NhaS is going to be activated. There were chosen UV rays as the initiator of the NhaS expression because they are present in normal conditions, since the mentioned UV promoter is activated under 360 wavelengths of light spectrum. The following table shows the result of the promoter under a bright field and a fluorescence microscope observing the GFP protein reporter glowing, according to Colombian Team 2007.

Figure 7: Results of the BBa_I765001 UV promoter part according to Colombian Team 2007.

Other teams that used 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.

How to know there is any production?

In our ideal project we are going to propose an odor Wintergreen reporter for this module in order to know if there is any production of NhaS, that is our plan, but since Wintergreen has not been probed yet and NhaS is a putative protein we decided to test it 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 and obtaining conclusions about each one. Further information can be found in the Aroma module of our project.

Other teams that used NhaS and potential future uses

There are no teams that used this gene in the past. IGEM CIDEB 2014 is the first team that is going to synthesize, test and register it.

We consider this to be useful for the scientific community in general. 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 (1992), 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 Na+ /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.

NhaS part’s description

References

IVEY Mark, KRULWICH Terry. (1994) Sodium ion binding proteins. Retrieved April 1st 2014 from http://www.google.com.mx/patents/US5346815.

BMM Cancer Research UK. Interactive 3D Jigsaw. Retrieved May 1st 2014 from http://bmm.cancerresearchuk.org/.

Predict Protein. Request ID: 473277. Retrieved May 3rd 2014 from https://www.predictprotein.org

Yeast Resource Center. Protein Overview: gi|2209269. Retrieved May 1st 2014 from http://www.yeastrc.org/pdr/viewProtein.do?id=1296033

IGEM Colombian Team (2007) UV promoter. Retrieved April 1st 2014 from http://parts.igem.org/Part:BBa_I765001.

Antiquity (2013, January 31). Part: BBa_B0034. Retrieved May 1st 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034

Colombia Israel Team (2007) Part: BBa_I765001. Retrieved April 1st 2014 from http://parts.igem.org/Part:BBa_I765001

Knight Lab (2006) Part: BBa_B1002. Retrieved from http://parts.igem.org/Part:BBa_B1002