Team:Kyoto/Project
1) Motivation
Synthetic biology faces our daily challenges and aims to develop new devices that provide solutions to every situation, every environment, and every problem that exists in the world. Many iGEM teams try to create such devices. Some teams try to collect heavy metal from sea water, drainage and so on, and other teams aim to detect some substrates through protein connections. Do they work most effectively under their applied environment? Insufficient of the concentration will be solved by adding sodium. But how about when salt concentrations are too high? You may think adding water is fine, but how will you dilute sea or sewage? Can you do that? Adding water changes other ion concentrations as well. Once devices that absorb salt are developed in certain surrounding containing strong salt concentration, we can greatly support various functions of other devices for sensing of substances in the environment and bioremediation. Therefore we aim to create a yeast desalination system in order to make iGEMers’ devices work appropriately under their applied situation.
2) Intracellular ionic strength influences various life phenomena
Life phenomena occur in an aqueous solution system. Therefore, various properties of aqueous solutions that depend on ions have various effects on life phenomenon. For example, electrostatic shielding and influence on hydration water by ions will have an effect on ionic bonds, hydrophobic effects, van der Waals’ forces. This will impact on protein-protein interactions and enzyme reactions by changing the stability of nucleic acid molecules and proteins. The asymmetric distribution of ions between the outside and inside of biological membrane enables rapid reaction response of the living body by generating membrane potential, and the change of water potential due to ion influences the mechanical environment of the cell by changing its osmotic pressure. For this reason, almost all reactions in vivo, including RNA folding, DNA double-stranded structure, protein-protein interactions, are greatly affected by salt concentration in solution. In other words, the created tools of iGEMers using biomolecules are greatly affected by the salt concentration in the environment. However, the iGEMer and their devices are developed to solve every possible problem in the world, so it is often unavoidable to face harsh circumstances or face the need to reduce Na + concentration. Our aim is to gather Na + from solution in biological process and to reduce salt concentration so that we could help these tools do their best performance.
3) Living organism has various transporters that selectively permeate Na +
Na + is one of the most abundant and functionally utilized ions in living organisms, and organisms have many Na + transporters to control and use. For example, Na + / K + - ATPase converts the energy of ATP into an electrochemical gradient through Na + transport, which establishes mass exchangeability of living organisms by becoming a driving force for co-transport and counter transport. Na + can be stopped due to maintained internal and external membrane concentration difference. It controls the membrane potential and further establishes the electrical function of the organism by acting as a source of the action potential by the voltage-dependent channel. In addition, Na + controls the resting membrane potential and works as a source of action potential to establish the electrical function of organisms. Thus, while Na + widely affects chemical reactions in vivo, it is an element widely and extensively present in the environment, such as seawater, and therefore often causes a problem. Some organisms, such as salt plants growing in the brackish water area, are known to have systems for protecting cells from such high concentrations of salt.
4) Let's create a system that absorbs salt from a liquid using genetically modified yeast
In certain salt-type plants, they developed a system that sequesters Na + from the cytoplasm to vacuoles in order to protect themselves from Na + flowing in. In addition, some plants retain Na + tolerance system by producing a compatible solution. In the process of searching papers, we noticed that many genes involved in Na + sequestration also function in budding yeast and give salt tolerance. The budding yeast itself also maintains a mechanism for releasing Na + out of the cell, and it is also known that in some budding yeasts in which those genes have been destroyed and the mechanism failed, they are very sensitive to the salt contained in the medium. If these mechanisms can be combined, it is possible that a "biological desalination" device that stores Na + at a high concentration in the vacuoles of budding yeast and lowers the salt concentration of the extracellular fluid. We further investigated and we noticed that team Aachen 2017, created a similar budding yeast. We contacted the team Aachen and received a detailed story. Here we proposed a question to ourselves. Can we combine genes of salt plants and genes of salt-resistant soy sauce yeasts and build a desalination system of levels team Aachen did not reach? We started to create budding yeast expressing various genes, with "the device that minimizes the salt concentration in the medium in the test tube as much as possible" as the primary goal.
5) Aggregation system for raising the efficiency of recollection and biosafety
As mentioned above, we initially aimed to create a yeast that removes salt in "test tubes". As a result, we assumed to promote the usage of the biosensor, which detects trace elements contained in test samples such as blood and waste liquid, without being inhibited their function by salt in solution. However, from the activities at Human Practice, we have found a new goal that this system can also be used for social problems such as to stop the problem of salt damage. Assuming such a case, we have to incorporate a mechanism that prevents genetically modified yeast from diffusing into the environment. For this purpose, we tried introducing a new aggregation system as a system for collecting yeast more efficiently.
6) Calculating the amount of yeast necessary to lower the concentration through modeling
The advantage of a salt removal system with artificially placed defined components is that you can operate the amount and combination of these components freely. You can use different factors depending on the situation, such as the difference in initial salt concentration of an extracellular fluid, a difference in target salt concentration, a difference in time required for salt concentration manipulation, a difference in a required amount of yeast and more. In order to enable this flexible application, it is necessary that the system to be used is sufficiently understood, and modeling is done mathematically. Therefore, we constructed a model structurally from transporter dynamics and actually used that model to optimize our device. With this model, we were able to optimize our device in various environments that we could not demonstrate this time.