(Sea-)
A system developed to hunt and bind NaCl from seawater
To remove sodium and chloride from seawater, we planned for two different approaches; The first could be done by modifying microorganisms (yeasts) to tolerate, accumulate and lastly desalinate salty water.
The other one is depending on the use of proteins only with no need for any microorganisms at all, as proteins will be produced in vitro using cell-free expression systems. Lack of GMOs safety issues and more public acceptance are particular privileges to the second system only.
Accumulation Approach
This approach could be achieved through three major steps
● Increasing sodium influx in both the cell and vacuole
● Eliminating sodium efflux to the surrounding water
● Expressing osmoprotectants that improve cell survival under high salinity stress
We were inspired by two previous iGEM teams; Aachen 2017 and Kyoto 2018 who worked also on sodium removal from salty water.
Chassis
We picked Debaryomyces hansenii (yeast) as a chassis for several reasons:
1] It can accumulate high concentrations of sodium, without being intoxicated.
2] It is within the normal flora of sea-water.
3] It is halotolerant (it can tolerate salinity up to 4 M NaCl), Osmotolerant and Xerotolerant.
4] It is considered as biosafety level 1.
1.Sodium influx inside yeast cells
Sodium ions concentration in seawater is much more than that in yeast’s cytoplasm. Accordingly, sodium ions move by osmosis from the surrounding water to cytoplasm through non-selective ion channels. For further enhancement of this process, we designed a new part (BBa_K3144002) to express PHO89 transporter. The PHO89 gene code for high-affinity phosphate cotransporters of the plasma membrane which catalyzes a sodium-dependent phosphate uptake at alkaline pH, we specifically chose this part as it is highly induced by the little phosphate concentrations * and phosphate concentration in the seawater could assure high expression of this channel.
2.Sodium accumulation inside vacuoles
In order to remove sodium ions from the cytoplasm to prevent their toxic effects on a living cell, we decided to express SseNHX (BBa_K2665005) channel that codes for vacuolar Na+/H+ antiporter. SseNHX is a chimeric vacuolar Na+/H+ antiporter gene that maintains ionic homeostasis by sequestering excessive sodium into the vacuole* and designed as biobrick by Kyoto 2018 iGEM team.
As mentioned above, the NHX antiporter requires the presence of H+ to allow the passage of Na+ from cytoplasm to vacuole. So, increasing the H+ ions in the vacuole significantly enhances the accumulation process. This drives us to integrate AVP (chain-A pyrophosphate-energized vacuolar membrane proton pump) that acidifies vacuoles in plant cells and does not have a homologue in yeast.
3.Chloride influx inside vacuole
For chloride ions influx, we designed a new part (BBa_K3144003) to express OsCLC-1. This channel has voltage-gated chloride channel activity in Oryza sativa. OsCLC-1 plays a major role in the transport of chloride ions across the vacuolar membrane making O. Sativa tolerate saline environment and improve salt tolerance in GEF1 deficient yeast.
4.Stop sodium efflux
Halo-tolerant yeasts in general and Debaryomyces hansenii, in particular, has a strong efflux system to get rid of redundant sodium ions. To remove sodium and chloride ions from saline, we should prevent this opposing movement of ions. This could be done by knocking out the NHA1 gene that codes a plasma membrane Na+/ H+ antiporter which exports sodium ions from cell and takes up external protons in exchange for these internal sodium ions.
5.Yeast cells survival under salinity stress
After increasing sodium ions influx and eliminating its efflux, the yeast cell is now under very high salinity stress that could lead to its lysis as salt-induced stress results in two different phenomena: ion toxicity and osmotic stress. Cell lysis means sodium retrieval to the water and desalination will not take place. Accordingly, cells survival until being removed from the water is our first priority. We chose to overcome this by expressing some osmoprotectants and test how much they could increase salt tolerance of yeast. Osmoprotectants or compatible solutes are small organic molecules with neutral charge and low toxicity at high concentrations.
We designed new parts (BBa_K3144000 & BBa_K3144001) which introduce the myo-inositol pathway in Debaryomyces hansenii as it is not naturally expressed and it could improve its tolerance. We also decided to test and characterize another osmoprotectant which is Trehalose (BBa_K847060 and BBa_K847061) designed by (iGEM12_Stanford-Brown).
Cell-free Approach
For a more safe, acceptable and quick Biodesalination, we decided to desalinate water in a cell-free system. In this approach, we aimed to synthesize a chimeric sodium and chloride binding protein using different binding domains found in nature. So, we decided to use metal-binding proteins and test their affinities for sodium and chloride ions, knowing that the binding process is not element specific and mainly depends on the electrostatic attraction between the ions in the solution and the amino acid residues. For that reason, we depended on a big number of parts from the iGEM registry that were submitted previously as metal-binding proteins. (BBa_K231000, BBa_K643002, BBa_K643000, BBa_K1321005, BBa_K190020, BBa_K1701000, BBa_K1321138, BBa_K1321159, BBa_K1550010, BBa_K245129, BBa_K1613024, BBa_K1420004, BBa_K1980000, BBa_K1438001, BBa_K1460002, BBa_K1122702, BBa_K2665005 and BBa_K2225000)
The first step is to test their sodium binding affinities first in silico using Molecular dynamics web server I-tasser. Then in vitro using cell-free expression system (myTXTL® – Cell-Free Protein Expression – Arbor Biosciences), in order to do that we designed our system to be under the control of the constitutive family promoter member (BBa_J23102) and strong RBS (BBa_B0034), as strength of the latter highly affects the yield of protein production. Upon protein production, we aimed to test the affinity of the previously mentioned parts to bind sodium and chloride leading to reduction of TDS (Total Dissolved Salts) of water.
We planned to immobilize the best protein candidates on membranes so they could be easily removed from the water after desalination.
Hardware
Searching for an appropriate method to isolate yeasts from the water after the desalination process, we found some primitive ways; such as filtration, sifting, and evaporation. However, these ways were not sufficient to be used in the separation of the mixture as the yeast size is in micrometer; in addition, we were thinking for low power energy separation way for future works. This is why the hydrocyclone was the best choice for our design. The hydrocyclone is a static device that uses centrifugal force to separate heavy and light components from a liquid mixture; we used it as its design is sufficient to separate yeast from water. The mixture flows through the inlet tangentially, goes downwards in a spiral motion, thereby producing the centrifugal force. The larger particles go out through the spigot and the pure water is then lifted to the overflow outlet. It would be fabricated using 3D printing to customize its dimensions on our system.
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