1 Integrated Human Practice regarding future application

　In order to broaden the prospect for applications of our device, we conducted an interview with various people. Among them, the problem of salt damage was overwhelmingly rising from people's mouth like a big problem. Salt damage is caused by a large amount of salt remaining in cropland and we learned that this worldwide problem and even it is also common in Japan. Therefore, we started to think that we could propose a salt-sucking device as one of the solution methods for salt damage. We incorporated the opinions of experts, thought about how to use it even in and out of the lab environment i.e. in an actual field.

　1-1 About Salt damage
We contacted many of the salt damage experts and started human practice activity from learning about real-world problems caused by salt damage. Salt damage occurs in 1/5 of the world's agricultural land. In Japan as well, due to the tsunami associated with the Great East Japan Earthquake, 2.6% of the cultivated land area in the Tohoku region was affected. In Miyagi prefecture alone, 11% of the arable land area suffers from salt damage. In the world, food shortages are a concern because of the exponential growth of the human population. If effective agricultural land is lost due to salt damage, it is clearly that the food shortage situation will become more and more serious.

　　　　　　　　　　　　　　　　　　　　　　　　　Soil salinization caused by irrigation(Provided by Dr.Funakawa)

1-2 How to apply device to field
　Can our device perform desalination of problematic salt water in agricultural land? First, we asked Mr. Funukawa of Kyoto University who is familiar with the soil. (Link to Human Practice) In our initial thought, we were thinking of removing all salt from the area where salt damage is occurring and making it an agricultural land where crops can be raised once again. However, Mr. Funekawa insisted that "It is not realistic to have the yeast to absorb it because about 1 t / ha of salt is accumulated on farmland where salt damage is chronically occurring." Based on his advice, we reached a conclusion that application of our device will be limited to 'farmland where high concentrations of salt begin to accumulate', not to 'redeem farmland from complete salt damage'. In other words, we decided to prevent the problem of salt damage, not to resolve a problem which already occurred. Also, Professor Funekawa said, "Some of the farmers who are fighting against salt damage dilute the wastewater that sucked out the salt in the soil with fresh water and then reuse it again" If we could combine it with our device, we can reduce the salt concentration of the wastewater, and it can be reused as leaching water once again. By doing this, we expect that we could possibly carry out desalination work without worrying about water resources, and it will become possible to work more effectively against salt damage more and more. We decided to use our device to desalinate waste water.

　1-3 Where to apply device
　As explained in the Human Practice, freshwater is abundant in Japan, so the opportunity to use our device domestically was considered to be very limited (link). However, salt damage is not only a problem in Japan, but it is also a problem to be considered on a global scale. We thought that somewhere in the world there might be places that need such a desalination system so we decided to talk with a researcher who knows a lot about salt damage occurring overseas.
　First of all, we asked Mr. Ishida who has been studying salt damage for years while conducting field surveys on desertification and salt damage in the Aral Sea. According to his story, salt damage causes damage not only to agricultural crops but also causes direct influence on the human body so salt damage solution in arid lands was found to be a very important task. Also, from his story we learned that the amount of river water is insufficient, such that the water of Aral Sea is depleted. We considered that our system, which has the feature of conserving water, would be effective for dry grounds.

　　　　　　　　　　　　　　　　　　　　　Deteriorated soil in arid land(provided by Dr.Funakawa)

　Next, we asked Mr. Fujimaki of Tottori University about the applicability of our system to the dry land. "In the plains of Iraq and Egypt, which have been facing salt damage from ancient times, draining facilities are pretty popular, and we can recollect nearly 100% of wastewater when it does not rain heavily." We confirmed that there is a possibility that our yeast system for desalination from wastewater can be installed in the dry areas of Middle East area such as Iraq and Egypt.

　　　　　　　　　　　　　　　　　　　　　Drainage in Egypt (provided by Dr.Fujimaki)

Through expert opinions and mentorship, we were able to learn about the problem of salt damage and extend the project's purpose and contribution to society.

2 Integrated Human Practice regarding biosafety

We were only thinking about biological and technical aspects with respect to creating a device using yeast that suck salt. Initially, we only considered the condition inside the test tube as a place to use our device, and considering biosafety was sufficient in order to adhere to laboratory safety guidelines. However, as we wrote in the above item, the policy has changed to expand the purpose of our device and aim for future use in the field of salt damage. In this case, unlike laboratory work, it is necessary to give special consideration so as not to let genetically modified yeast escape into the environment.

Bureau frequently pointed out that whether we are taking genetically modified organisms go outside the system and harm human beings or have a negative impact on the ecosystem. When applying to out-laboratory environments (salt damage problems) it is necessary to give special consideration so as not to let genetically modified yeast escape into the environment. We noticed that we must be more carefully about biosafety.

　2-1 column fixing yeasts
　So we started thinking the importance to set up biosafety. As we talked to Professor Inoue about yeast, he introduced us a method to fix yeast to the column. If yeast is immobilized on a column and a solution to be desalted is passed through the column, solution can be desalted without mixing with the yeast cells. With this method, the solution after desalting should be safely desalted without including yeast cells.
　In addition to that, we decided to fix yeasts in the column that are aggregated beforehand, in order to further enhance the biosafety. This reduces the number of yeast cells that exist alone and prevents small yeast cells from coming out of the column.
　Aggregation of yeasts can be performed by expressing two proteins that bind together specifically; SdrG and Fgb, which are said to be the most potent in the living world.

2-2 completed desalination system 　We tried to solve the environmental problems of salt damage by fixing salt-absorbing yeast on a column and applying it to an practical field. As a result, we finally reached the following desalination system.
　First, we supposed to install our system on mild salt-damage farmlands where agriculture is somehow being carried out, to try to prevent further salt accumulation.Then, wastewater containing salt will be collected through drainage equipments such as underdrains, passed through a column fixing yeasts, and desalinated so that drainage can be reused as clean water. In addition, we chose dry areas for installation of our system. This is because serious salt damage occurs in irrigated agriculture in arid areas, and our system, which can coscerve water ,also attribute to alleviation of water shortage in arid areas.

　3 Integrated Human Practice regarding progress of project

Similar to the circumstances of reconfiguring project objectives and consideration of safety equipment discussed above, dialogue with a number of experts was involved in the progress of our laboratory work. In what follows, we will show how our project has changed by feedback from each expert.

　　3-1 Zygosaccharomyces rouxii
　Professor Watanabe, a specialist in soy sauce yeast, taught us various important information about Zygosaccharomyces rouxii. As we mentioned in Human Practice section, the direct reason why we introduced ZrGPD1 and ZrFPS1 gene in parts used for subsequent devices is because we confirmed the salt-tolerance of soy sauce yeast by ourselves based on the information from professor Watanabe and our experiments.
　We give grate thanks to Professor Watanabe's lecture because without it couldn’t culture soy sauce yeast nor analysis genome information.

　　3-2 mutant strain of yeast
　Mr.Uozumi is an expert who has analyzed salt tolerance by introducing plant genes into budding yeast. We got advice from Mr. Uozumi to select the budding yeast mutant needed for our device. As a result, we got a clue on the yeast strain to be used and additionally gave us the strain G19, which is a yeast mutant that absorbs the salt very well. Although this G19 strain could not be used directly for our device due to the limited number selective markers, we aimed to create yeast mutants with has similar genotype by ourselves with the aim of the making the similar type of strain G19.

　3-3 SLiCE
　If we could construct plasmids and introduce mutations into genes more easily and cheaply......... This is the dream of all iGEMers. Our experiments often lead to dead end this summer, and we continued to explore other possibilities. We encountered with the SLiCE method in such a situation. (Please refer to the link for detailed protocols. ) As a result of introducing SLiCE this time, we succeeded in cloning in some of the genes that were difficult with other cloning methods.
　We thought that such a low-cost cloning method would be beneficial for a student in iGEM. We would like to disseminate the SLiCE method to other teams of iGEM. We already talked about the SLiCE method when we interacted with the Gifu team. Since we are introducing it on our wiki (link), please try SLiCE methods for other iGEM teams as well. (Linked to collaboration)

　3-4 boil method
　When we started the yeast project that absorbs salt, the past iGEM project that we referred to most was Aachen 2017. In this project, the possibility of absorbing salt into yeast was clearly introduced, but there was no clear information about how much salt could actually be absorbed by yeast.
　In order to create a yeast that absorbs Na + ions from the outside, measuring Na + ion concentration accurately and rapidly is most important. From the beginning of the project, finding a method to measure ion has always been a concern of our team.
　We send an email to all possible researchers and finally, we succeeded in obtaining cooperation from Mr.Kobayashi and Mrs. Ochiai of Kyoto University, which is the biggest factor that made this research possible. This two expert devised a boil method as a method of measuring Na + in yeast, and with the advent of boil method and measuring instrument, it became possible to accurately measure the concentration of sodium ions in a cell. It would not have been possible to do an experimental assay without the cooperation of the two.

　Add-up
　As introduced above, our project of this year has been achieved with the cooperation of many people. Thanks to the expert interview which summarized in the item on salt damage section, we were able to greatly extend the scope of our intended purpose of use from in-vitro use to future application with salt damage. Also, thanks to many experts introduced in the "Project" section, we were able to construct many plasmids and to evaluate the performance of actual device prototypes. Furthermore, last but not least, the serious interest in biosafety drawn from many experts, high school students and the general public changed our thoughts radically and add a new concept to our device.

Is your HP issues has been integrated into the purpose, design, and/or execution of your project ?

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　From https://2018.igem.org/Human_Practices/How_to_Succeed
　We have……
　learn about the specific circumstance of environmental problem “salt damage” through expert interview and successfully designed a new system.
　recognized the needs of further safety measures for our devices through education & public engagment, and we developed new parts to showcase more biosafty..
　groped a experimental method that enables us to do our labolatory work even easir and accurately through expert interview.