Aachen

　Aachen 2017 was doing a project to recover Na + from factory wastewater. Its design was close to our objective and there were data to be helpful in many respects. We contacted them, taught their unpublished data, and got advice on our experimental plan. Please see the link for details : Aachen 2017

Knock-out-strain

　Initially, we were planning to make ΔENA1 strain, ΔNHA1 strain and ΔENA1ΔNHA1 double knocked out strain with reference to team Aachen’s previous report that ΔENA1 and ΔNHA1 strain frequently take in salt. Then when we were consulting with Prof. Uozumi, about one salt resistance gene (HKT 1), we learned to use a strain named G19 (ΔENA1-4) which expects to have greater ability to absorb salt. Experiments with this strain revealed that the amount of salt absorption was quite large, but G19 strain could not be used because of the limited number of the marker. So we made our own standard ΔENA1,2,5 knockout yeast which lacks same corresponding gene as ENA1-4.

Soy sauce yeast

　We were thinking of what should be good for salt resistance genes to put into yeast. As we looked through many papers, it turned out that soy sauce yeast is a promising candidate. So I went to ask Dr. Watanabe of Japanese soy sauce maker Yamasa Soy Sauce. Dr. Watanabe taught the experimental method of dealing with soy sauce yeast and also introduced us a few soy sauce yeast strains with salt tolerance. As a result of using the soy sauce yeast, we found that it shows very strong salt tolerance, and the effectiveness of the experiment to introduce the resistance gene of soy sauce yeast into budding yeast was supported.

　It turned out that using soy sauce yeast genes to create seasonings that we normally use is effective for obtaining salt tolerance!

Boil Method

　We had to investigate how much salt was absorbed by the yeast. Initially, we were thinking about using sodium measurement drugs like Thermo Fisher 's Corona Green and requiring measuring external companies to measure, but they were expensive and could not afford to use it. When we looked at a laboratory that likely has an instrument for measuring ions in the laboratory at Kyoto University, I found that Dr.Kobayashi of the agriculture department had been measuring ion concentration. We decided to use the atomic absorption spectrophotometer using there.
　A method of measuring the sodium ion concentration in the cells, crushing cells and extracting the contents was first proposed, but by strong recommendation of Dr.Kobayashi, we decided to suspend the yeast in distilled water after boiling and centrifuge supernatant was taken. And by taking out and diluting to certain amount and measure; Boiling method. Special Methods
　It became possible to actually measure sodium ion concentration!

Column method

　In order to learn more about yeast itself, we went to see Dr.Inoue who is specializing in yeast. We held a discussion based on this year’s project outline. As the discussion progressed, we faced a problem of biosafety and as an answer to the problem, he proposed to implement a device using a column. The design of this device is like this: first, we will fix yeast to a specific matrix using a classical method called immobilization bacteria. Then we will fill that in a cylinder. The advantage of this device is that there is no worry about the leakage of yeast in this case. We got a big hint on how to safely use our device.

　SLiCE method

　We are trying to develop devices that can help other synthetic biological devices and contribute to social problems including salt damage. In reality, however, most of the work we did was to prepare plasmids and create knockout yeast strains. In order to efficiently conduct original research, it is indispensable to establish a system that makes this plasmid construction easily, reliably and quickly. A literature search was conducted while struggling with PCR and gBlock, and as one way to solve this problem, SLiCE method was introduced.
　 　SLiCE method is a seamless cloning method which was originally developed by Zhang et al in 2012. This method was further optimized by Prof. Motohashi of Kyoto Sangyo University. The feature of the optimized method is that it is low cost and protocol is easy for students to reproduce. We suppose that such a low-cost cloning method would be beneficial for all students participating in iGEM. However, as we looked for information on the internet, there were pros and cons evenly so we could not assure if this method is reliable or not. Fortunately, Prof. Motohashi who developed this method was in Kyoto, so we decided to ask in person and establish the protocol of SLiCE method.

In fact, SLiCE was a very simple method. As it is mentioned in the result section, there was an example of cloning which succeeded for the first time by the method of SLiCE. From the above, we believe that this method is one of the dreamlike ways to make cloning work easier. In the same way, it is expected that we can share this method with other iGEMers suffering from cloning and release all iGEMers from the difficulty of the work.