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Desalination

The Concept

We sought to develop a strain of yeast that would have the ability to desalinate sea water enough to result in potable drinking water. Our goal was to modify the yeast ion regulation system such that it would take in much higher than normal amounts of sodium and sequester it within its vacuoles. This posed a serious problem, as biological systems will naturally work to maintain a low internal concentration of sodium ions, while concentrating potassium ions within the cytoplasm. Because of their like charges and relative size, these elements compete with each other for protein interaction within the cell, and because potassium is much more important for biological processes and much less available in the natural environment, yeast actively avoids accumulation of sodium and works to concentrate potassium within itself.

Wild-type strains of yeast have innumerable methods of maintaining the correct internal balance. We chose to focus our efforts on the regulation of ions across the plasma and vacuole membranes, by manipulating several transmembrane ion pumps. In the presence of high salinity environments, yeast will generally react in the following ways:

1) Decrease the flow of ions into the cell.
2) Increase the flow of ions out of the cell.
3) Increase the flow of ions into the intracellular compartments.

In order to maximize the efficiency of desalination, we sought to eliminate its ability to stop the inflow of ions (1). In fact, we wanted to increase the inflow when in high salinity environments. The team also chose to eliminate the main pumps responsible for (2). We created a deletion strain for these genes. And lastly, we wanted to enhance the organism's ability for (3).

The Theoretical Mechanism

The following is a list of genes that we sought to modify in some way

A) NHA1: codes for an Na⁺/H⁺ antiporter
An endogenous high capacity sodium transporter located in the plasmas membrane, which removes sodium from the cytoplasm in exchange for protons, and ejects them back into the environment. We desired to relocate this to the vacuolar membrane. The effect should be the same: ions would still be removed from the cytoplasm, maintaining the proper concentrations necessary to prevent cell death.

B) NHX1: codes for a Na⁺/H⁺ antiporter
Another endogenous sodium transporter. This one is naturally present in the endosomal transport system, particularly in the vacuole membrane. This pump is much less selective than NHA1p and thus has a lower efficiency, which is why we chose to relocate that protein, even with the presence of this antiporter. We did choose to over-express it, however, in order to ensure an even more efficient means for the sequestration of sodium ions.

C) ENA1,2&5: code for Na⁺ATPases, with varying efficiencies
These are endogenous sodium ATPases, whose sole purpose is to eliminate sodium from the cell, especially in high salinity situations. When in the presence of large concentrations of sodium, these genes are upregulated, and the cell becomes very efficient at removing large amounts of sodium from within the cytoplasm. We chose to completely remove this capability, with the knowledge that this could very well result in cell death. We believe that, with our other modifications, we can compensate for what would otherwise be lethal levels of sodium. This is the deletion strain we created, to which we plan to add the other modifications.

D) ChR2/ChEF: code for light-activate channelrhodopsin
A nonendogenous (?) light activated ion channel. We thought that adding this to the plasma membrane would be a novel mechanism for increasing our yeast's ability to draw in sodium. It would also allow us "turn on" and "turn off" an increased sodium uptake. The channelrhodopsin opens at specific wavelengths, allowing for the free movement of ions across the plasma membrane. as long as sodium is continuously being moved from the cytoplasm to the vacuole, the ion concentration with the cytoplasm would remain low enough to maintain a large gradient across the membrane.

http://mmbr.asm.org/content/74/1/95/F1.large.jpg

Results

Our plan was to design our bio-bricks using Gibson Assembly and gBlocks. However we were unable to get the Gibson Assemblies to work, and we were unable to produce our parts. One big hurdle in designing our parts was that some of the sequences for the proteins we wanted to use had many restriction enzyme sites that were not compatible with the bio-brick standards. This is why we chose Gibson Assembly of gBlocks over PCRing our sequences out of the yeast genome.

We did some early-on preliminary desalination testing of the wild-type strains of S. cerevisiae, with some promising results. It was able to naturally remove a significant amount of salt from the water. We created salt water at the same concentration as would be found in sea water using NaCl. We had the option of using artificial sea water from on-campus salt water tanks, but thought that the various other ions and minerals would interfere with our initial data collection. Stay tuned for quantitative data results!

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