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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 system, particularly in the vauole 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<sup>+
C) a third part was creating a yeast strain with all plasma membrane Na- ATPases (ENA1, 2 and 5)knocked out, preventing the yeast from expelling the yeast back into the environment, a normal wildtype behavior

D) and the fourth part was to design a channel rhodopsin that would allow the system to be light activated, only allowing Na to enter the cell when exposed to a specific wave length of light.</sup>

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 were 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.