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Team:CSU Fort Collins/Desalination
From 2013.igem.org
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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.
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. so that it would be redirected from the plasma membrane to the yeast vacuole
B) a second part involved the modification of an endogenous Na transporter (NHX1) present on the vacuole so that it would be over expressed, enhancing the rate of sodium movement
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.
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.
